Humanized anti-FGF19 antagonists and methods using same

ABSTRACT

The present invention concerns antagonists of the FGF19/FGFR4 pathways, and the uses of same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Patent ApplicationNo. PCT/US2008/071955, filed Aug. 1, 2008, and claims priority under 35USC §119 to U.S. Provisional Patent Application No. 60/953,908, filedAug. 3, 2007, the entire contents of which are hereby incorporated byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 29, 2010, isnamed P4012R1.txt and is 59,682 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology. More specifically, the invention concerns antagonists of theFGF19/FGFR4 pathways, and the uses of same.

BACKGROUND OF THE INVENTION

The fibroblast growth factor (FGF) family is composed of 22 structurallyrelated polypeptides that bind to 4 receptor tyrosine kinases (FGFR1-4)and one kinase deficient receptor (FGFR5) (Eswarakumar et al (2005)Cytokine Growth Factor Rev 16, 139-149; Ornitz et al (2001) Genome Biol2, REVIEWS3005; Sleeman et al (2001) Gene 271, 171-182). FGFs'interaction with FGFR1-4 results in receptor homodimerization andautophosphorylation, recruitment of cytosolic adaptors such as FRS2 andinitiation of multiple signaling pathways (Powers et al (2000) EndocrRelat Cancer 7, 165-197; Schlessinger, J. (2004) Science 306,1506-1507).

FGFs and FGFRs play important roles in development and tissue repair byregulating cell proliferation, migration, chemotaxis, differentiation,morphogenesis and angiogenesis (Ornitz et al (2001) Genome Biol 2,REVIEWS3005; Auguste et al (2003) Cell Tissue Res 314, 157-166; Steilinget al (2003) Curr Opin Biotechnol 14, 533-537). Several FGFs and FGFRsare associated with the pathogenesis of breast, prostate, cervix,stomach and colon cancers (Jeffers et al (2002) Expert Opin Ther Targets6, 469-482; Mattila et al. (2001) Oncogene 20, 2791-2804; Ruohola et al.(2001) Cancer Res 61, 4229-4237; Marsh et al (1999) Oncogene 18,1053-1060; Shimokawa et al (2003) Cancer Res 63, 6116-6120; Jang (2001)Cancer Res 61, 3541-3543; Cappellen (1999) Nat Genet. 23, 18-20;Gowardhan (2005) Br J Cancer 92, 320-327).

FGF19 is a member of the most distant of the seven subfamilies of theFGFs. FGF19 is a high affinity ligand of FGFR4 (Xie et al (1999)Cytokine 11:729-735). FGF19 is normally secreted by the biliary andintestinal epithelium. FGF19 plays a role in cholesterol homeostasis byrepressing hepatic expression of cholesterol-7-α-hydroxylase 1 (Cyp7α1),the rate-limiting enzyme for cholesterol and bile acid synthesis(Gutierrez et al (2006) Arterioscler Thromb Vasc Biol 26, 301-306; Yu etal (2000) JBiol Chem 275, 15482-15489; Holt, JA, et al. (2003) Genes Dev17(130):158). FGF19 ectopic expression in a transgenic mouse modelincreases hepatocytes proliferation, promotes hepatocellular dysplasiaand results in neoplasia by 10 months of age (Nicholes et al. (2002). AmJ Pathol 160, 2295-2307). The mechanism of FGF19 induced hepatocellularcarcinoma is thought to involve FGFR4 interaction. Treatment with FGF-19increases metabolic rate and reverses dietary and leptin-deficientdiabetes. Fu et al (2004) Endocrinology 145:2594-2603. FGF-19 is alsodescribed in, for example, Xie et al. (1999) Cytokine 11:729-735; Harmeret al (2004) Biochemistry 43:629-640; Desnoyer, LR et al, Oncogene(2008) 27(1): 85-97; and Lin, BC et al. (2007) J Biol Chem282(37):27277-84; Pai, R et al. Cancer Res (2008) 68(13):5086-95.

FGFR4 expression is widely distributed and was reported in developingskeletal muscles, liver, lung, pancreas, adrenal, kidney and brain (Kanet al. (1999) J Biol Chem 274, 15947-15952; Nicholes et al. (2002) Am JPathol 160, 2295-2307; Ozawa et al. (1996) Brain Res Mol Brain Res 41,279-288; Stark et al (1991) Development 113, 641-651). FGFR4amplification was reported in mammary and ovarian adenocarcinomas(Jaakkola et al (1993) Int J Cancer 54, 378-382). FGFR4 mutation andtruncation were correlated with the malignancy and in some cases theprognosis of prostate and lung adenocarcinomas, head and neck squamouscell carcinoma, soft tissue sarcoma, astrocytoma and pituitary adenomas(Jaakkola et al (1993) Int J Cancer 54, 378-382; Morimoto (2003) Cancer98, 2245-2250; Qian (2004) J Clin Endocrinol Metab 89, 1904-1911;Spinola et al. (2005) J Clin Oncol 23, 7307-7311; Streit et al (2004)Int J Cancer 111, 213-217; Wang (1994) Mol Cell Biol 14, 181-188; Yamada(2002) Neurol Res 24, 244-248).

It is clear that there continues to be a need for agents that haveclinical attributes that are optimal for development as therapeuticagents. The invention described herein meets this need and providesother benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention is in part based on the identification of a variety ofantagonists of the FGF19/FGFR4 pathway. FGF19 presents as an importantand advantageous therapeutic target, and the invention providescompositions and methods based on interfering with FGF19/FGFR4activation, including but not limited to interfering with FGF19 bindingto FGFR4 extracellular domain. Antagonists of the invention, asdescribed herein, provide important therapeutic and diagnostic agentsfor use in targeting pathological conditions associated with expressionand/or activity of the FGF19-FGFR4 pathway. Accordingly, the inventionprovides methods, compositions, kits and articles of manufacture relatedto modulating the FGF19/FGFR4 pathway, including modulation of FGF19receptor binding, activation, and other biological/physiologicalactivities associated with FGF19/FGFR4 signaling.

For example, in one embodiment, the invention provides a humanizedanti-FGF19 antibody wherein the monovalent affinity of the antibody tohuman FGF19 (e.g., affinity of the antibody as a Fab fragment to humanFGF19) is substantially the same as the monovalent affinity of a murineantibody (e.g., affinity of the murine antibody as a Fab fragment tohuman FGF19) or a chimeric antibody (e.g., affinity of the chimericantibody as a Fab fragment to human FGF19) comprising, consisting orconsisting essentially of a light chain and heavy chain variable domainsequence as depicted in FIG. 8. In another embodiment, the inventionprovides a humanized anti-FGF19 antibody wherein the monovalent affinityof the antibody to human FGF19 (e.g., affinity of the antibody as a Fabfragment to human FGF19) is lower, for example at least 3, 5, 7 or10-fold lower, than the monovalent affinity of a murine antibody (e.g.,affinity of the murine antibody as a Fab fragment to human FGF19) or achimeric antibody (e.g., affinity of the chimeric antibody as a Fabfragment to human FGF19) comprising, consisting or consistingessentially of a light chain and heavy chain variable domain sequence asdepicted in FIG. 8. In another embodiment, the invention provides ananti-FGF19 humanized antibody wherein the monovalent affinity of theantibody to human FGF19 (e.g., affinity of the antibody as a Fabfragment to human FGF19) is greater, for example at least 3, 5, 7, 10 or13-fold greater, than the monovalent affinity of a murine antibody(e.g., affinity of the murine antibody as a Fab fragment to human FGF19)or a chimeric antibody (e.g., affinity of the chimeric antibody as a Fabfragment to human FGF19) comprising, consisting or consistingessentially of a light chain and heavy chain variable domain sequence asdepicted in FIG. 8. As is well-established in the art, binding affinityof a ligand to its receptor can be determined using any of a variety ofassays, and expressed in terms of a variety of quantitative values.Accordingly, in one embodiment, the binding affinity is expressed as Kdvalues and reflects intrinsic binding affinity (e.g., with minimizedavidity effects). Generally and preferably, binding affinity is measuredin vitro, whether in a cell-free or cell-associated setting. Asdescribed in greater detail herein, fold difference in binding affinitycan be quantified in terms of the ratio of the monovalent bindingaffinity value of a humanized antibody (e.g., in Fab form) and themonovalent binding affinity value of a reference/comparator antibody(e.g., in Fab form) (e.g., a murine antibody having donor hypervariableregion sequences), wherein the binding affinity values are determinedunder similar assay conditions. Thus, in one embodiment, the folddifference in binding affinity is determined as the ratio of the Kdvalues of the humanized antibody in Fab form and saidreference/comparator Fab antibody. For example, in one embodiment, if anantibody of the invention (A) has an affinity that is “3-fold lower”than the affinity of a reference antibody (M), then if the Kd value forA is 3×, the Kd value of M would be 1×, and the ratio of Kd of A to Kdof M would be 3:1. Conversely, in one embodiment, if an antibody of theinvention (C) has an affinity that is “3-fold greater” than the affinityof a reference antibody (R), then if the Kd value for C is 1×, the Kdvalue of R would be 3×, and the ratio of Kd of C to Kd of R would be1:3. Any of a number of assays known in the art, including thosedescribed herein, can be used to obtain binding affinity measurements,including, for example, Biacore, radioimmunoassay (RIA) and ELISA.

In one aspect, a FGF19 antagonist of the invention comprises ananti-FGF19 antibody comprising:

(a) at least one, two, three, four or five hypervariable region (HVR)sequences selected from the group consisting of:

(i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is KASQDINSFLS(SEQ ID NO:1)

(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is RANRLVD (SEQ IDNO:2)

(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is LQYDEFPLT (SEQID NO:3)

(iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GFSLTTYGVH(SEQ ID NO:4)

(v) HVR-H2 comprising sequence E1-E17, wherein E1-E17 isXVIWPGGGTDYNAAFIS (SEQ ID NO:5) and X is not G, and

(vi) HVR-H3 comprising sequence F1-F13, wherein F1-F13 is XXKEYANLYAMDY(SEQ ID NO:6) and X at position F1 is not V and X at position F2 is notR;

and (b) at least one (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)variant HVR,

wherein the variant HVR sequence comprises modification of at least oneresidue of the sequence depicted in SEQ ID NOs:1, 2, 3, 4, 5 or 6. Inone embodiment, HVR-L1 of an antibody of the invention comprises thesequence of SEQ ID NO:1. In one embodiment, HVR-L2 of an antibody of theinvention comprises the sequence of SEQ ID NO:2. In one embodiment,HVR-L3 of an antibody of the invention comprises the sequence of SEQ IDNO:3. In one embodiment, HVR-H1 of an antibody of the inventioncomprises the sequence of SEQ ID NO:4. In one embodiment, HVR-H2 of anantibody of the invention comprises the sequence of SEQ ID NO:5. In oneembodiment, HVR-H3 of an antibody of the invention comprises thesequence of SEQ ID NO:6. In one embodiment, HVR-H2 comprisesGVIWPGGGTDYNAAFIS (SEQ ID NO: 7). In one embodiment, HVR-H3 comprisesVRKEYANLYAMDY (SEQ ID NO: 8). In one embodiment, HVR-H3 comprisesVXKEYANLYAMDY (SEQ ID NO: 9), wherein X is not R. In one embodiment,HVR-H3 comprises XRKEYANLYAMDY (SEQ ID NO: 10), wherein X is not V. Inone embodiment, HVR-L1 comprises KASQDINSFLA (SEQ ID NO: 11). In oneembodiment, HVR-L1 comprises KASQDINSFLG (SEQ ID NO: 12). In oneembodiment, HVR-L2 comprises RANRLVS (SEQ ID NO: 13). In one embodiment,HVR-L2 comprises RANRLVE (SEQ ID NO: 14). In one embodiment, an antibodyof the invention comprising these sequences (in combination as describedherein) is humanized or human. These antibodies are distinct from (i.e.they are not) an antibody described in U.S. patent application Ser. No.11/673,411, filed Feb. 9, 2007.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or six HVRs, wherein each HVR comprises, consists orconsists essentially of a sequence selected from the group consisting ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 14 and whereinSEQ ID NO:1, 11 and 12 correspond to an HVR-L1, SEQ ID NO:2, 13, and 14correspond to an HVR-L2, SEQ ID NO:3 corresponds to an HVR-L3, SEQ IDNO:4 corresponds to an HVR-H1, SEQ ID NO:5 or 7 correspond to an HVR-H2,and SEQ ID NOs:6, 8, 9 or 10 correspond to an HVR-H3. In one embodiment,an antibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:1, 2, 3,4, 7 and 8. In one embodiment, an antibody of the invention comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises SEQ ID NO:1, 2, 3, 4, 7 and 9. In one embodiment, anantibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:1, 2, 3,4, 7 and 10. In one embodiment, an antibody of the invention comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises SEQ ID NO:1, 11, 3, 4, 7 and 8. In one embodiment, anantibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:1, 12,3, 4, 7 and 8. In one embodiment, an antibody of the invention comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises SEQ ID NO:1, 11, 13, 4, 7 and 8. In one embodiment, anantibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:1, 12,13, 4, 7 and 8. In one embodiment, an antibody of the inventioncomprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, whereineach, in order, comprises SEQ ID NO:1, 11, 14, 4, 7 and 8. In oneembodiment, an antibody of the invention comprises HVR-L1, HVR-L2,HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order, comprisesSEQ ID NO:1, 12, 14, 4, 7 and 8. In one embodiment, an antibody of theinvention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3,wherein each, in order, comprises SEQ ID NO:1, 11, 13, 4, 7 and 9. Inone embodiment, an antibody of the invention comprises HVR-L1, HVR-L2,HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, in order, comprisesSEQ ID NO:1, 11, 13, 4, 7 and 10. In some embodiments, these antibodiesfurther comprise a human subgroup III heavy chain framework consensussequence. In some embodiment of these antibodies, these antibodiesfurther comprise a human κI light chain framework consensus sequence.

Variant HVRs in an antibody of the invention can have modifications ofone or more residues within the HVR.

In one embodiment, a HVR-L1 variant comprises 1-11 (1, 2, 3, 4, 5, 6, 7,8, 9, 10 or 11) substitutions in any combination of the followingpositions: A1 (T, S, Q or N), A2 (V, S, L or P), A3 (V, Y, I, R, N, K orQ), A4 (E, L, H, K, R or S), A5 (H, N, G, R, E or Y), A6 (F, L, A, V, K,S or M), A7 (M, K, D, Y or I), A8 (N, A, K, R, Y or I), A9 (S, Y or L),A10 (M, V or I), and A11 (A, G or T).

In one embodiment, a HVR-L2 variant comprises 1-7 (1, 2, 3, 4, 5, 6 or7) substitutions in any combination of the following positions: B1 (K,G, T, S, Q or H), B2 (T, G or S), B3 (K, S, G, Y, R, E or I), B4 (M, G,Y, H or L), B5 (Q, M, V, I or H), B6 (E, R, M, A, G or P), and B7 (E, A,V, N or G).

In one embodiment, a HVR-L3 variant comprises 1-8 (1, 2, 3, 4, 5, 6, 7or 8) substitutions in any combination of the following positions: C1 (Mor Q), C2 (S, T, N, K, H, E, D or A), C3 (D or F), C4 (S, A, E, G, H, Y,N or V), C5 (G, K, T, D, N, V, Y, A or I), C6 (M), C7 (A), and C9 (S, Ior V).

In one embodiment, a HVR-H1 variant comprises 1-8 (1, 2, 3, 4, 5, 6, 7or 8) substitutions in any combination of the following positions: D2(Y), D3 (R, G, N or D), D4 (I, V, F or M), D5 (A, I, K, N, R or S), D6(S or R), D7 (F), D9 (A or G) and D10 (Q or Y).

In one embodiment, a HVR-H2 variant comprises 1-14 (1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13 or 14) substitutions in any combination of thefollowing positions: E1 (S), E2 (I, L or M), E3 (S, M, V, L, F, Y or T),E6 (A), E8 (A, T, S, Y or R), E9 (I, L, S, V or Y), E10 (E, N, A or H),E11 (E, S, L, F, I, V or W), E12 (G, K, A, T or S), E13 (E, K, S, G, Por T), E14 (R, E, L, G, F, D, T, S or K), E15 (L, V or S), E16 (T, E, N,L, S, V, M, A, T, H, G, D or F), and E17 (T, R, N, G, K, P, A, D or E).

In one embodiment, a HVR-H3 variant comprises 1-9 (1, 2, 3, 4, 5, 6, 7,8 or 9) substitutions in any combination of the following positions: F4(A, G, K or Q), F6 (G), F7 (S, K, T or F), F8 (V or I), F9 F, S or G),F10 (R, K, Q, E, L, M, P, T or V), F11 (L, F, A or S), F12 (T, H, E, N,V, A, Q or Y) and F13 (H, F, N or S).

Letter(s) in parenthesis following each position indicates anillustrative substitution (i.e., replacement) amino acid; as would beevident to one skilled in the art, suitability of other amino acids assubstitution amino acids in the context described herein can beroutinely assessed using techniques known in the art and/or describedherein. In one embodiment, A11 in a variant HVR-L1 is T. In oneembodiment, A11 in a variant HVR-L1 is A. In one embodiment, B7 in avariant HVR-L2 is S. In one embodiment, B7 in a variant HVR-L2 is G. Inone embodiment, A11 in a variant HVR-L1 is T and B7 in a variant HVR-L2is S. In one embodiment, A11 in a variant HVR-L1 is T and B7 in avariant HVR-L2 is G. In one embodiment, A11 in a variant HVR-L1 is A andB7 in a variant HVR-L2 is S. In one embodiment, A11 in a variant HVR-L1is T and B7 in a variant HVR-L2 is G. In one embodiment, D9 in a variantHVR-H1 is A. In one embodiment, D10 in a variant HVR-H1 is Q. In oneembodiment, E2 in a variant HVR-H2 is L. In one embodiment, F10 in avariant HVR-H3 is R. In one embodiment, F10 in a variant HVR-H3 is R andF11 in the variant HVR-H3 is S.

In some embodiments, these antibodies further comprise a human subgroupIII heavy chain framework consensus sequence. In some embodiment ofthese antibodies, these antibodies further comprise a human KI lightchain framework consensus sequence.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or all of the HVR sequences depicted in FIG. 3 (SEQ IDNOs:18 and 52-260).

A therapeutic agent for use in a host subject preferably elicits littleto no immunogenic response against the agent in said subject. In oneembodiment, the invention provides such an agent. For example, in oneembodiment, the invention provides a humanized antibody that elicitsand/or is expected to elicit a human anti-mouse antibody response (HAMA)at a substantially reduced level compared to an antibody comprising theheavy and light chain variable regions shown in FIG. 8 in a hostsubject. In another example, the invention provides a humanized antibodythat elicits and/or is expected to elicit minimal or no human anti-mouseantibody response (HAMA). In one example, an antibody of the inventionelicits anti-mouse antibody response that is at or less than aclinically-acceptable level.

As is known in the art, and as described in greater detail hereinbelow,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below). The invention provides antibodies comprisingmodifications in these hybrid hypervariable positions.

An antibody of the invention can comprise any suitable human or humanconsensus light chain framework sequences, provided the antibodyexhibits the desired biological characteristics (e.g., a desired bindingaffinity). In some embodiments, one or more (such as 2, 3, 4, 5, 6, 7,8, 9, or more) additional modifications are present within the humanand/or human consensus non-hypervariable region sequences. In oneembodiment, an antibody of the invention comprises at least a portion(or all) of the framework sequence of human κ light chain. In oneembodiment, an antibody of the invention comprises at least a portion(or all) of human κ subgroup I framework consensus sequence. In someembodiments, antibodies of the invention comprise a human subgroup IIIheavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73 and/or 78. In some embodiments of these antibodies,position 71 is A, 73 is T and/or 78 is A. In one embodiment, an antibodyof the invention comprises a heavy and/or light chain variable domaincomprising framework sequence depicted in FIG. 1 and/or FIG. 2, providedposition 49 in the heavy chain is not G and/or position 93 in the heavychain is not V and/or position 94 in the heavy chain is not R.

Some embodiments of antibodies of the invention comprise a light chainvariable domain of humanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN®,Genentech, Inc., South San Francisco, CA, USA) (also referred to in U.S.Pat. No. 6,407,213 and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93)as depicted in SEQ ID NO:269 below.

(SEQ ID NO: 269) 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala  Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

 Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(HVR residues are underlined)In one embodiment, the huMAb4D5-8light chain variable domain sequence ismodified at one or more of positions 30, 66 and 91 (Asn, Arg and His asindicated in bold/italics above, respectively). In one embodiment, themodified huMAb4D5-8 sequence comprises Ser in position 30, Gly inposition 66 and/or Ser in position 91. Accordingly, in one embodiment,an antibody of the invention comprises a light chain variable domaincomprising the sequence depicted in SEQ ID NO:15 below:

(SEQ ID NO: 15) 1 Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys AlaPro Lys Leu Leu Ile Tyr Ser Ala Ser Phe LeuTyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

 Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(HVR residues are underlined)Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics above.

In one aspect, an antibody of the invention is a humanized anti-FGF19antibody that inhibits binding of human FGF19 to FGFR4 substantially thesame as a reference antibody (such as a chimeric anti-FGF19 antibody ora murine anti-FGF19 antibody) comprising a light chain and heavy chainvariable sequence as depicted in FIG. 8. Comparison of abilities toinhibit FGF19 binding to its receptor can be performed according tovarious methods known in the art, including as described in the Examplesbelow. In one embodiment, IC50 values are determined across an antibodyconcentration range from about 0.01 nM to around 1000 nM.

In one aspect, an antibody of the invention is a humanized anti-FGF19antibody that inhibits human FGFR4 receptor activation substantially thesame as a reference antibody (such as a chimeric anti-FGF19 antibody ora murine anti-FGF19 antibody) comprising a light chain and heavy chainvariable sequence as depicted in FIG. 7 (SEQ ID NO: 9 and 10).Comparison of abilities to inhibit receptor activation can be performedaccording to various methods known in the art, including as described inthe Examples below. In one embodiment, IC50 values are determined acrossan antibody concentration range from about 0.1 nM to about 100 nM.

In one embodiment, both the humanized antibody and chimeric antibody aremonovalent. In one embodiment, the reference chimeric antibody comprisesvariable domain sequences depicted in FIG. 8 linked to a human Fcregion. In one embodiment, the human Fc region is that of an IgG (e.g.,IgG1, 2, 3 or 4).

In one aspect, the invention provides an anti-FGF19 antibody comprising:at least one, two, three, four, five, and/or six hypervariable region(HVR) sequences selected from the group consisting of: (a) HVR-L1comprising the sequence depicted in SEQ ID NO:1; (b) HVR-L2 comprisingthe sequence depicted in SEQ ID NO:11; (c) HVR-L3 comprising thesequence depicted in SEQ ID NO:13; (d) HVR-H1 comprising the sequencedepicted in SEQ ID NO:4; (e) HVR-H2 comprising the sequence depicted inSEQ ID NO:7; and (f) HVR-H3 comprising the sequence depicted in SEQ IDNO:8.

In one aspect, the invention provides an anti-FGF19 antibody comprisinga light chain comprising (a) HVR-L1 comprising the sequence depicted inSEQ ID NO: 1; (b) HVR-L2 comprising the sequence depicted in SEQ IDNO:11; and (c) HVR-L3 comprising the sequence depicted in SEQ ID NO:13.

In one aspect, the invention provides an anti-FGF19 antibody comprisinga heavy chain comprising (a) HVR-H1 comprising the sequence depicted inSEQ ID NO:4; (b) HVR-H2 comprising the sequence depicted in SEQ ID NO:7;and (c) HVR-H3 comprising the sequence depicted in SEQ ID NO:8.

In one aspect, the invention provides an anti-FGF19 antibody comprising(a) a light chain comprising (i) HVR-L1 comprising the sequence depictedin SEQ ID NO:1; (ii) HVR-L2 comprising the sequence depicted in SEQ IDNO:11; and (iii) HVR-L3 comprising the sequence depicted in SEQ IDNO:13, and (b) a heavy chain comprising (i) HVR-H1 comprising thesequence depicted in SEQ ID NO:4; (ii) HVR-H2 comprising the sequencedepicted in SEQ ID NO:7; and (iii) HVR-H3 comprising the sequencedepicted in SEQ ID NO:8.

In some embodiments, these antibodies further comprise a human subgroupIII heavy chain framework consensus sequence. In some embodiment ofthese antibodies, these antibodies further comprise a human KI lightchain framework consensus sequence.

The antibodies of the invention may modulate one or more aspects ofFGF19- and FGFR4-associated effects, including but not limited to FGF19binding, FGFR4 activation, FGFR4 downstream molecular signaling,disruption of FGFR4 binding to FGF19, FGFR4 multimerization, expressionof a CYP7α1 gene, phosphorylation of FGFR4, MAPK, FRS2 and/or ERK2,activation of β-catenin, FGF19-promoted cell migration, and/ordisruption of any biologically relevant FGF19 and/or FGFR4 biologicalpathway, and/or treatment and/or prevention of a tumor, cellproliferative disorder or a cancer; and/or treatment or prevention of adisorder associated with FGF19 expression and/or activity (such asincreased FGF19 expression and/or activity).

In some embodiments, the antibody of the invention specifically binds toFGF19. In some embodiments, the antibody specifically binds FGF19 with aKd of about 120 μM or stronger. In some embodiments, the antibodyspecifically binds FGF19 with a Kd of about 140 pM or stronger. In someembodiments, the antibody blocks FGF19 binding to FGFR4 with an IC50 ofabout 4 nM.

In one aspect, the invention provides an isolated antibody that binds anFGFR4 binding region of FGF19.

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks FGF19-induced repression ofexpression of a CYP7α1 gene in a cell exposed to FGF19.

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks FGF19-induced phosphorylation ofFGFR4, MAPK, FRS2 and/or ERK2 in a cell exposed to FGF19.

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks FGF19-promoted cell migration. Insome embodiments, the cell is a tumor cell. In some embodiments, thecell is a tumor cell. In some embodiments, the cell is an HCT116 cell.

In one aspect, the invention provides an isolated anti-FGF19 antibodythat inhibits, reduces, and/or blocks Wnt pathway activation in a cell.In some embodiments, Wnt pathway activation comprises one or more ofβ-catenin immunoreactivity, tyrosine phosphorylation of β-catenin,expression of Wnt target genes, β-catenin mutation, and E-cadherinbinding to β-catenin. Detection of Wnt pathway activation is known inthe art, and some examples are described and exemplified herein.

In one embodiment, an antibody of the invention specifically binds toFGF19 of a first animal species, and does not specifically bind to FGF19of a second animal species. In one embodiment, the first animal speciesis human and/or primate (e.g., cynomolgus monkey), and the second animalspecies is murine (e.g., mouse) and/or canine. In one embodiment, thefirst animal species is human. In one embodiment, the first animalspecies is primate, for example cynomolgus monkey. In one embodiment,the second animal species is murine, for example mouse. In oneembodiment, the second animal species is canine.

In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a polyclonal antibody. In some embodiments,the antibody is selected from the group consisting of a chimericantibody, an affinity matured antibody, a humanized antibody, and ahuman antibody. In some embodiments, the antibody is an antibodyfragment. In some embodiments, the antibody is a Fab, Fab′, Fab′-SH,F(ab′)₂, or scFv.

In one aspect, the invention provides an antibody that competes with anyof the above-mentioned antibodies for binding to FGF19 (i.e., blocksbinding to FGF19 of any of the above-mentioned antibodies). In oneaspect, the invention provides an antibody that binds to the sameepitope on FGF19 as any of the above-mentioned antibodies.

In other embodiments, the antibodies of the invention further comprisechanges in amino acid residues in the Fc region that lead to improvedeffector function including enhanced CDC and/or ADCC function and B-cellkilling. Other antibodies of the invention include those having specificchanges that improve stability. In other embodiments, the antibodies ofthe invention comprise changes in amino acid residues in the Fc regionthat lead to decreased effector function, e.g. decreased CDC and/or ADCCfunction and/or decreased B-cell killing. In some embodiments, theantibodies of the invention are characterized by decreased binding (suchas absence of binding) to human complement factor C1q and/or human Fcreceptor on natural killer (NK) cells. In some embodiments, theantibodies of the invention are characterized by decreased binding (suchas the absence of binding) to human FcγRI, FcγRIIA, and/or FcγRIIIA. Insome embodiments, the antibodies of the invention is of the IgG class(e.g., IgG1 or IgG4) and comprises at least one mutation in E233, L234,L235, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and/orP329 (numbering according to the EU index). In some embodiments, theantibodies comprise the mutation L234A/L235A or D265A/N297A.

In one aspect, the invention provides anti-FGF19 polypeptides comprisingany of the antigen binding sequences provided herein, wherein theanti-FGF19 polypeptides specifically bind to FGF19.

In one aspect, the invention provides an immunoconjugate(interchangeably termed “antibody drug conjugate” or “ADC”) comprisingany of the anti-FGF19 antibodies disclosed herein conjugated to anagent, such as a drug.

In one aspect, the invention provides compositions comprising one ormore antibodies of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In another aspect, the invention supplies a composition comprising oneor more anti-FGF19 antibodies described herein, and a carrier. Thiscomposition may further comprise a second medicament, wherein theantibody is a first medicament. This second medicament, for cancertreatment, for example, may be another antibody, chemotherapeutic agent,cytotoxic agent, anti-angiogenic agent, immunosuppressive agent,prodrug, cytokine, cytokine antagonist, cytotoxic radiotherapy,corticosteroid, anti-emetic cancer vaccine, analgesic, anti-vascularagent, or growth-inhibitory agent. In another embodiment, a secondmedicament is administered to the subject in an effective amount,wherein the antibody is a first medicament. This second medicament ismore than one medicament, and is preferably another antibody,chemotherapeutic agent, cytotoxic agent, anti-angiogenic agent,immunosuppressive agent, prodrug, cytokine, cytokine antagonist,cytotoxic radiotherapy, corticosteroid, anti-emetic, cancer vaccine,analgesic, anti-vascular agent, or growth-inhibitory agent. Morespecific agents include, for example, irinotecan (CAMPTOSAR®), cetuximab(ERBITUX®), fulvestrant (FASLODEX®), vinorelbine (NAVELBINEO),EFG-receptor antagonists such as erlotinib (TARCEVA®) VEGF antagonistssuch as bevacizumab (AVASTIN®), vincristine (ONCOVIN®), inhibitors ofmTor (a serine/threonine protein kinase) such as rapamycin and CCI-779,and anti-HER1, HER2, ErbB, and/or EGFR antagonists such as trastuzumab(HERCEPTIN®), pertuzumab (OMNITARG™), or lapatinib, and other cytotoxicagents including chemotherapeutic agents. In some embodiments, thesecond medicament is an anti-estrogen drug such as tamoxifen,fulvestrant, or an aromatase inhibitor, an antagonist to vascularendothelial growth factor (VEGF) or to ErbB or the Efb receptor, orHer-1 or Her-2. In some embodiments, the second medicament is tamoxifen,letrozole, exemestane, anastrozole, irinotecan, cetuximab, fulvestrant,vinorelbine, erlotinib, bevacizumab, vincristine, imatinib, sorafenib,lapatinib, or trastuzumab, and preferably, the second medicament iserlotinib, bevacizumab, or trastuzumab.

In one aspect, the invention provides an anti-idiotype antibody thatspecifically binds an anti-FGF19 antibody of the invention.

In one aspect, the invention provides nucleic acids encoding ananti-FGF19 antibody of the invention.

In one aspect, the invention provides vectors comprising a nucleic acidof the invention.

In one aspect, the invention provides compositions comprising one ormore nucleic acid of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In one embodiment, a host cellis a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In one aspect, the invention provides methods of making an antibody ofthe invention. For example, the invention provides methods of making ananti-FGF19 antibody (which, as defined herein includes full length andfragments thereof), said method comprising expressing in a suitable hostcell a recombinant vector of the invention encoding said antibody, andrecovering said antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more anti-FGF19antibodies of the invention. In one embodiment, the compositioncomprises a nucleic acid of the invention. In one embodiment, acomposition comprising an antibody further comprises a carrier, which insome embodiments is pharmaceutically acceptable. In one embodiment, anarticle of manufacture of the invention further comprises instructionsfor administering the composition (for e.g., the antibody) to anindividual (such as instructions for any of the methods describedherein).

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more anti-FGF19 antibodies ofthe invention; and a second container comprising a buffer. In oneembodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antibody further comprises acarrier, which in some embodiments is pharmaceutically acceptable. Inone embodiment, a kit further comprises instructions for administeringthe composition (for e.g., the antibody) to an individual.

In one aspect, the invention provides use of an anti-FGF19 antibody ofthe invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disorder, such as a cancer, a tumor,and/or a cell proliferative disorder. In some embodiments, the cancer, atumor, and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the cancer, a tumor,and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the cancer, a tumor,and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disorder, such as a cancer, a tumor, and/ora cell proliferative disorder. In some embodiments, the cancer, a tumor,and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disorder, such as a cancer, a tumor,and/or a cell proliferative disorder. In some embodiments, the cancer, atumor, and/or a cell proliferative disorder is colorectal cancer,hepatocellular carcinoma, lung cancer, breast cancer, or pancreaticcancer. In some embodiments, the disorder is a liver disorder, such ascirrhosis. In some embodiments, the disorder is a wasting disorder.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disorder, such as a cancer, a tumor, and/or a cellproliferative disorder. In some embodiments, the cancer, a tumor, and/ora cell proliferative disorder is colorectal cancer, hepatocellularcarcinoma, lung cancer, breast cancer, or pancreatic cancer. In someembodiments, the disorder is a liver disorder, such as cirrhosis. Insome embodiments, the disorder is a wasting disorder.

The invention provides methods and compositions useful for modulating adisease associated with dysregulation of the FGF19/FGFR4 signaling axis(such as modulating disease states associated with expression and/oractivity of FGF19 and/or FGFR4), said methods comprising administrationof an effective dose of an anti-FGF19 antibody to an individual in needof such treatment.

In one aspect, the invention provides methods for killing a cell (suchas a cancer or tumor cell), the methods comprising administering aneffective amount of an anti-FGF19 antibody to an individual in need ofsuch treatment.

In one aspect, the invention provides methods for reducing, inhibiting,blocking, or preventing growth of a tumor or cancer, the methodscomprising administering an effective amount of an anti-FGF19 antibodyto an individual in need of such treatment.

Methods of the invention can be used to affect any suitable pathologicalstate. Exemplary disorders are described herein, and include a cancerselected from the group consisting of esophageal cancer, bladder cancer,lung cancer, ovarian cancer, pancreatic cancer, mammary fibroadenoma,prostate cancer, head and neck squamous cell carcinoma, soft tissuesarcoma, astrocytoma, pituitary cancer, breast cancer, neuroblastomas,melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC),epithelial carcinomas, brain cancer, endometrial cancer, testis cancer,cholangiocarcinoma, gallbladder carcinoma, and hepatocellular carcinoma.

In one embodiment, a cell that is targeted in a method of the inventionis a cancer cell. For example, a cancer cell can be one selected fromthe group consisting of a breast cancer cell, a colorectal cancer cell,a lung cancer cell, a papillary carcinoma cell, a colon cancer cell, apancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell,a central nervous system cancer cell, an esophageal cancer cell, anosteogenic sarcoma cell, a renal carcinoma cell, a hepatocellularcarcinoma cell, a bladder cancer cell, a gastric carcinoma cell, a headand neck squamous carcinoma cell, a melanoma cell, a leukemia cell, abrain cancer cell, a endometrial cancer cell, a testis cancer cell, acholangiocarcinoma cell, a gallbladder carcinoma cell, a lung cancercell, and/or a prostate cancer cell. In one embodiment, a cell that istargeted in a method of the invention is a hyperproliferative and/orhyperplastic cell. In one embodiment, a cell that is targeted in amethod of the invention is a dysplastic cell. In yet another embodiment,a cell that is targeted in a method of the invention is a metastaticcell.

In one embodiment of the invention, the cell that is targeted is acirrhotic liver cell.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (for e.g., a cancer cell) isexposed to radiation treatment or a chemotherapeutic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: depicts alignment of sequences of the variable light chain forthe following: light chain human κI consensus sequence (SEQ ID NO:261),murine 1A6 antibody (SEQ ID NO:17) and, the 1A6 grafted antibody (SEQ IDNO:262). Positions are numbered according to Kabat.

FIG. 2: depicts alignment of sequences of the variable heavy chain forthe following: light chain variable heavy subgroup III consensussequence (SEQ ID NO:263), murine 1A6 antibody (SEQ ID NO:16) and the 1A6grafted antibody (SEQ ID NO:264). Positions are numbered according toKabat.

FIGS. 3A-D: depicts various HVR sequences of selected affinity-maturedantibodies from libraries with individually-randomized HVR. HVR-L1: SEQID NOS: 1, 18 and 52-86; HVR-L2: SEQ ID NOS: 2, 87-127; HVR-L3: SEQ IDNOS: 3, 128-155; HVR-H1: SEQ ID NOS: 4, 156-176; HVR-H2: SEQ ID NOS: 7,177-229; AND HVR=H3: SEQ ID NOS: 8, 230-260.

FIGS. 4A,B & 5: depict exemplary acceptor human consensus frameworksequences for use in practicing the instant invention with sequenceidentifiers as follows:

Variable heavy (VH) consensus frameworks (FIG. 4A, B)

-   human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NOS    19-21 & 49, respectively, in order of appearance)-   human VH subgroup I consensus framework minus extended hypervariable    regions (SEQ ID NOS 22-23, 21 & 49; 22-24 & 49; and 22-23, 25 & 49,    respectively, in order of appearance)-   human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID    NOS 26-28 & 49, respectively, in order of appearance)-   human VH subgroup II consensus framework minus extended    hypervariable regions (SEQ ID N0S 29-30, 28 & 49; 29-31 & 49; and    29-30, 32 & 49, respectively, in order of appearance)-   human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID    NOS 33-35 & 49, respectively, in order of appearance)-   human VH subgroup III consensus framework minus extended    hypervariable regions (SEQ ID NOS 46-47, 35 & 49; 46-47, 36 & 49;    and 46-49, respectively, in order of appearance)-   human VH acceptor framework minus Kabat CDRs (SEQ ID NOS 37, 34, 268    & 49, respectively, in order of appearance)-   human VH acceptor framework minus extended hypervariable regions    (SEQ ID NOS 46-47, 268 & 49 and 46-47, 267 & 49, respectively, in    order of appearance)-   human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NOS 37, 34,    266 & 49, respectively, in order of appearance)-   human VH acceptor 2 framework minus extended hypervariable regions    (SEQ ID NOS 46-47, 266 & 49; 46-47, 265 & 49; and 46-47, 270 & 49,    respectively, in order of appearance)

Variable light (VL) consensus frameworks (FIG. 5)

-   human VL kappa subgroup I consensus framework (SEQ ID NOS 42-45,    respectively, in order of appearance)-   human VL kappa subgroup II consensus framework (SEQ ID NOS 38-40 &    45, respectively, in order of appearance)-   human VL kappa subgroup III consensus framework (SEQ ID NOS 41,    272-273 & 45, respectively, in order of appearance)-   human VL kappa subgroup IV consensus framework (SEQ ID NOS 274-276 &    45, respectively, in order of appearance)

FIG. 6: depicts framework region sequences of huMAb4D5-8 light and heavychains. Numbers in superscript/bold indicate amino acid positionsaccording to Kabat.

FIG. 7: depicts modified/variant framework region sequences ofhuMAb4D5-8 light and heavy chains. Numbers in superscript/bold indicateamino acid positions according to Kabat.

FIG. 8: depicts donor (murine antibody 1A6) light chain (LC) and heavychain (HC) variable domain sequences.

FIG. 9: Humanized anti-FGF19 antibody 1A6.v1 (“h1A6”) and chimericanti-FGF19 antibody 1A6 (“ch1A6”) demonstrated similar blockingactivity. In a solid phase receptor binding assay, hu1A6 and ch1A6blocked FGF19 interaction with FGFR4 with the same efficacy (IC50=4.5nM).

FIG. 10: Western blot analysis of FGF19 expression in human andcynomolgus liver. (A) Humanized anti-FGF19 antibody 1A6.v1 (“hu1A6”)bound to human and cynomolgus FGF19. (B) Humanized anti-FGF19 antibody1A6.v1 recognized recombinant huFGF19, recombinant cynoFGF19 andcynoFGF19 proteins isolated from the liver.

FIG. 11: Treatment with humanized anti-FGF10 antibody 1A6.v1 inhibitedFGFR4, FRS2 and ERK phosphorylation in vitro. Phosphorylation of FGFR4,FRS2, and ERK was inhibited in humanized anti-FGF19 antibody1A6.v1-treated HCT116 colon tumor cell.

FIG. 12: Treatment with humanized anti-FGF19 antibody 1A6.v1 inhibitedcolon tumor cell line growth in vivo. (A) Growth of HCT116 colon tumorxenografts was significantly inhibited by treatment with 30 mg/kg of1A6.v1 compared to control antibody (p=0.042). A 44% inhibition of tumorgrowth was observed when animals were treated with 30 mg/kg of 1A6.v1.(B) Phosphorylation of FGFR4, FRS2, and ERK was inhibited in humanizedanti-FGF19 antibody 1A6.v1-treated HCT116 xenograft tumors.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods, compositions, kits and articles ofmanufacture for identifying and/or using inhibitors of the FGF19/FGFR4signaling pathway.

Details of these methods, compositions, kits and articles of manufactureare provided herein.

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

Definitions

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “anti-FGF19 antibody” or “an antibody that binds to FGF19”refers to an antibody that is capable of binding FGF19 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting FGF19. Preferably, the extent of bindingof an anti-FGF19 antibody to an unrelated, non-FGF19 protein is lessthan about 10% of the binding of the antibody to FGF19 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to FGF19 has a dissociation constant (Kd) of ≦1 μM, ≦100 nM,≦10 nM, ≦1 nM, or ≦0.1 nM. In certain embodiments, an anti-FGF19antibody binds to an epitope of FGF19 that is conserved among FGF19 fromdifferent species.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 ug/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbant plate (Nunc #269620), 100 μMor 26 pM [¹²⁵I]-antigen antigen are mixed with serial dilutions of a Fabof interest (e.g., consistent with assessment of an anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., 65 hours) to insure that equilibriumis reached. Thereafter, the mixtures are transferred to the captureplate for incubation at room temperature (e.g., for one hour). Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 ul/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25 C with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol Biol 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilizedantigen CM5 chips at ˜10 response units (RU). Briefly, carboxymethylateddextran biosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (˜0.2uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. MolBiol 293:865-881. However, if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate is preferablydetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and a basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The term “FGF19” (interchangeably termed “Fibroblast growth factor 19”),as used herein, refers, unless specifically or contextually indicatedotherwise, to any native or variant (whether native or synthetic) FGF19polypeptide. The term “native sequence” specifically encompassesnaturally occurring truncated or secreted forms (e.g., an extracellulardomain sequence), naturally occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants. The term “wildtype FGF19” generally refers to a polypeptide comprising the amino acidsequence of a naturally occurring FGF19 protein. The term “wild typeFGF19 sequence” generally refers to an amino acid sequence found in anaturally occurring FGF19.

The term “FGFR4” (interchangeably termed “Fibroblast growth factorreceptor 4”), as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) FGFR4 polypeptide. The term “native sequence”specifically encompasses naturally occurring truncated or secreted forms(e.g., an extracellular domain sequence), naturally occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants. The term “wild type FGFR4” generally refers to apolypeptide comprising the amino acid sequence of a naturally occurringFGFR4 protein. The term “wild type FGFR4 sequence” generally refers toan amino acid sequence found in a naturally occurring FGFR4.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (HVRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three HVRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The HVRs in eachchain are held together in close proximity by the FR regions and, withthe HVRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In one embodiment, an antibody fragment comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, an antibody fragment, for example onethat comprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcRn binding, antibody half life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For e.g., such an antibodyfragment may comprise on antigen binding arm linked to an Fc sequencecapable of conferring in vivo stability to the fragment.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH(H1, H2, H3), and three in the VL (L1,L2, L3). In native antibodies, H3 and L3 display the most diversity ofthe six HVRs, and H3 in particular is believed to play a unique role inconferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo etal., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993). Triabodies and tetrabodies are alsodescribed in Hudson et al., Nat. Med. 9:129-134 (2003).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenic;challenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see U.S. Provisional Application No. 60/640,323, Figures for EUnumbering).

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies may be produced using certainprocedures known in the art. For example, Marks et al. Bio/Technology10:779-783 (1992) describes affinity maturation by VH and VL domainshuffling. Random mutagenesis of HVR and/or framework residues isdescribed by, for example, Barbas et al. Proc Nat. Acad. Sci. USA91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton etal. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896(1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which partially orfully mimics at least one of the functional activities of a polypeptideof interest.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays as disclosed, for example, in definitions herein.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daëron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie etal., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol.Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

Binding to human FcRn in vivo and serum half life of human FcRn highaffinity binding polypeptides can be assayed, e.g., in transgenic miceor transfected human cell lines expressing human FcRn, or in primates towhich the polypeptides with a variant Fc region are administered. WO2000/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al. J. Biol.Chem. 9(2):6591-6604 (2001).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils. The effector cells may be isolated from a native source,e.g., from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. NK cells, neutrophils, andmacrophages) enable these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta), may be performed. Useful effector cells for suchassays include PBMC and NK cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., inan animal model such as that disclosed in Clynes et al. PNAS (USA)95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising antibody” refers to an antibody thatcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the antibody or by recombinant engineering of thenucleic acid encoding the antibody. Accordingly, a compositioncomprising an antibody having an Fc region according to this inventioncan comprise an antibody with K447, with all K447 removed, or a mixtureof antibodies with and without the K447 residue.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework, or from a human consensus framework.An acceptor human framework “derived from” a human immunoglobulinframework or human consensus framework may comprise the same amino acidsequence thereof, or may contain pre-existing amino acid sequencechanges. Where pre-existing amino acid changes are present, preferablyno more than 5 and preferably 4 or less, or 3 or less, pre-existingamino acid changes are present. Where pre-existing amino acid changesare present in a VH, preferably those changes are only at three, two orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may be 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences:

EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 46)- H1-WVRQAPGKGLEWV(SEQ ID NO: 47)- H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 48)-H3-WGQGTLVTVSS. (SEQ ID NO: 49)

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 42)- DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 43)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 44)- L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 45) L3-FGQGTKVEIK.

A “biological sample” (interchangeably termed “sample” or “tissue orcell sample”) encompasses a variety of sample types obtained from anindividual and can be used in a diagnostic or monitoring assay. Thedefinition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The sourceof the biological sample may be solid tissue as from a fresh, frozenand/or preserved organ or tissue sample or biopsy or aspirate; blood orany blood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, peritoneal fluid, or interstitial fluid; cells from anytime in gestation or development of the subject. In some embodiments,the biological sample is obtained from a primary or metastatic tumor.The biological sample may contain compounds which are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

For the purposes herein a “section” of a tissue sample is meant a singlepart or piece of a tissue sample, e.g. a thin slice of tissue or cellscut from a tissue sample. It is understood that multiple sections oftissue samples may be taken and subjected to analysis according to thepresent invention. In some embodiments, the same section of tissuesample is analyzed at both morphological and molecular levels, or isanalyzed with respect to both protein and nucleic acid.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable.

A “medicament” is an active drug to treat the disorder in question orits symptoms, or side effects.

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors; carcinoma, blastoma, and sarcoma.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, pituitary cancer, esophageal cancer,astrocytoma, soft tissue sarcoma, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer,testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastriccancer, melanoma, and various types of head and neck cancer.Dysregulation of angiogenesis can lead to many disorders that can betreated by compositions and methods of the invention. These disordersinclude both non-neoplastic and neoplastic conditions. Neoplasticsinclude but are not limited those described above. Non-neoplasticdisorders include but are not limited to undesired or aberranthypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriaticplaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabeticand other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis (OA), refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), hemophilic joints, hypertrophic scars, inhibition of hairgrowth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

The term “wasting” disorders (e.g., wasting syndrome, cachexia,sarcopenia) refers to a disorder caused by undesirable and/or unhealthyloss of weight or loss of body cell mass. In the elderly as well as inAIDS and cancer patients, wasting disease can result in undesired lossof body weight, including both the fat and the fat-free compartments.Wasting diseases can be the result of inadequate intake of food and/ormetabolic changes related to illness and/or the aging process. Cancerpatients and AIDS patients, as well as patients following extensivesurgery or having chronic infections, immunologic diseases,hyperthyroidism, Crohn's disease, psychogenic disease, chronic heartfailure or other severe trauma, frequently suffer from wasting diseasewhich is sometimes also referred to as cachexia, a metabolic and,sometimes, an eating disorder. Cachexia is additionally characterized byhypermetabolism and hypercatabolism. Although cachexia and wastingdisease are frequently used interchangeably to refer to wastingconditions, there is at least one body of research which differentiatescachexia from wasting syndrome as a loss of fat-free mass, andparticularly, body cell mass (Mayer, 1999, J. Nutr. 129(1SSuppl.):2565-2595). Sarcopenia, yet another such disorder which canaffect the aging individual, is typically characterized by loss ofmuscle mass. End stage wasting disease as described above can develop inindividuals suffering from either cachexia or sarcopenia.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Forexample, an anti-angiogenesis agent is an antibody or other antagonistto an angiogenic agent as defined above, e.g., antibodies to VEGF,antibodies to VEGF receptors, small molecules that block VEGF receptorsignaling (e.g., PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinibmalate), AMG706). Anti-angiogensis agents also include nativeangiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g.,Klagsbrun and D'Amore, Annu Rev. Physiol., 53:217-39 (1991); Streit andDetmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo,Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene,22:6549-6556 (2003) (e.g., Table 2 listing antiangiogenic factors); and,Sato Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 listsAnti-angiogenic agents used in clinical trials).

An “individual,” “subject,” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, farm animals (such as cows), sport animals, pets (such ascats, dogs, and horses), primates, mice and rats. In certainembodiments, a mammal is a human.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “toxin” is any substance capable of having a detrimental effect on thegrowth or proliferation of a cell.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®,Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib),proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bc1-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors (see definition below);serine-threonine kinase inhibitors such as rapamycin (sirolimus,RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,SARASAR™); and pharmaceutically acceptable salts, acids or derivativesof any of the above; as well as combinations of two or more of the abovesuch as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; lutenizing hormone-releasing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and tripterelin; sex steroids, including progestines such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretionic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingFGF19) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one which significantly reduces the percentage of cells (such as acell expressing FGF19) in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in Mendelsohn and Israel, eds., TheMolecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W.B. Saunders,Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel)are anticancer drugs both derived from the yew tree. Docetaxel(TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is asemisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb).Paclitaxel and docetaxel promote the assembly of microtubules fromtubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the polypeptide or by recombinant engineering thenucleic acid encoding the polypeptide. Accordingly, a compositioncomprising a polypeptide having an Fc region according to this inventioncan comprise polypeptides with K447, with all K447 removed, or a mixtureof polypeptides with and without the K447 residue.

Generating Variant Antibodies Exhibiting Reduced or Absence of HAMAResponse

Reduction or elimination of a HAMA response is a significant aspect ofclinical development of suitable therapeutic agents. See, e.g.,Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al.,Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985),135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller etal., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352;Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res.(1990), 50:1495. As described herein, the invention provides antibodiesthat are humanized such that HAMA response is reduced or eliminated.Variants of these antibodies can further be obtained using routinemethods known in the art, some of which are further described below.

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or hypervariable sequence(s). A selected frameworksequence to which a starting hypervariable sequence is linked isreferred to herein as an acceptor human framework. While the acceptorhuman frameworks may be from, or derived from, a human immunoglobulin(the VL and/or VH regions thereof), preferably the acceptor humanframeworks are from, or derived from, a human consensus frameworksequence as such frameworks have been demonstrated to have minimal, orno, immunogenicity in human patients.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence that is selected based onits homology to the donor framework sequence by aligning the donorframework sequence with various human framework sequences in acollection of human framework sequences, and select the most homologousframework sequence as the acceptor.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup III and/or VL kappa subgroup I consensusframework sequences.

Thus, the VH acceptor human framework may comprise one, two, three orall of the following framework sequences:

FR1 comprising EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:46),

FR2 comprising WVRQAPGKGLEWV (SEQ ID NO:47),

FR3 comprising FR3 comprises RFTISX1DX2SKNTX3YLQMNSLRAEDTAVYYC (SEQ IDNO:50), wherein X1 is A or R, X2 is T or N, and X3 is A or L,

FR4 comprising WGQGTLVTVSS (SEQ ID NO:49).

Examples of VH consensus frameworks include:

human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NOS19-21 & 49, respectively, in order of appearance)

human VH subgroup I consensus framework minus extended hypervariableregions (SEQ ID NOS 22-23, 21 & 49; 22-24 & 49; and 22-23, 25 & 49,respectively, in order of appearance)

human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NOS26-28 & 49, respectively, in order of appearance)

human VH subgroup II consensus framework minus extended hypervariableregions (SEQ ID NOS 29-30, 28 & 49; 29-31 & 49; and 29-30, 32 & 49,respectively, in order of appearance)

human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID NOS33-35 & 49, respectively, in order of appearance)

human VH subgroup III consensus framework minus extended hypervariableregions (SEQ ID NOS 46-47, 35 & 49; 46-47, 36 & 49; and 46-48 & 49,respectively, in order of appearance)

human VH acceptor framework minus Kabat CDRs (SEQ ID NOS 37, 34, 268 &49, respectively, in order of appearance)

human VH acceptor framework minus extended hypervariable regions (SEQ IDN0S 46-47, 268 & 49and 46-47, 267 & 49, respectively, in order ofappearance)

human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NOS 37, 34, 266 &49, respectively, in order of appearance)

human VH acceptor 2 framework minus extended hypervariable regions (SEQID NOS 46-47, 266 & 49; 46-47, 265 & 49; and 46-47, 270 & 49,respectively, in order of appearance)

In one embodiment, the VH acceptor human framework comprises one, two,three or all of the following framework sequences:

FR1 comprising EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:46),

FR2 comprising WVRQAPGKGLEWV (SEQ ID NO:47),

FR3 comprising RFTISADTSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO:270),

-   RFTISADTSKNTAYLQMNSLRAEDTAVYYCA (SEQ ID NO:265),-   RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:266),

RFTISADTSKNTAYLQMNSLRAEDTAVYYCS (SEQ ID NO:267), or

RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (SEQ ID NO:268)

FR4 comprising WGQGTLVTVSS (SEQ ID NO:49).

The VL acceptor human framework may comprise one, two, three or all ofthe following framework sequences:

-   FR1 comprising DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:42),-   FR2 comprising WYQQKPGKAPKLLIY (SEQ ID NO:43),-   FR3 comprising GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:44),-   FR4 comprising FGQGTKVEIK (SEQ ID NO:45).-   Examples of VL consensus frameworks include:-   human VL kappa subgroup I consensus framework (SEQ ID NOS 42-45,    respectively, in order of appearance)-   human VL kappa subgroup II consensus framework (SEQ ID NOS 38-40 &    45, respectively, in order of appearance)-   human VL kappa subgroup III consensus framework (SEQ ID NOS 41,    272-273 & 45, respectively, in order of appearance) human VL kappa    subgroup IV consensus framework (SEQ ID NOS 274-276 & 45,    respectively, in order of appearance)

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that is from a human immunoglobulin or ahuman consensus framework, the present invention contemplates that theacceptor sequence may comprise pre-existing amino acid substitutionsrelative to the human immunoglobulin sequence or human consensusframework sequence. These pre-existing substitutions are preferablyminimal; usually four, three, two or one amino acid differences onlyrelative to the human immunoglobulin sequence or consensus frameworksequence.

Hypervariable region residues of the non-human antibody are incorporatedinto the VL and/or VH acceptor human frameworks. For example, one mayincorporate residues corresponding to the Kabat CDR residues, theChothia hypervariable loop residues, the Abm residues, and/or contactresidues. Optionally, the extended hypervariable region residues asfollows are incorporated: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35(H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3).

While “incorporation” of hypervariable region residues is discussedherein, it will be appreciated that this can be achieved in variousways, for example, nucleic acid encoding the desired amino acid sequencecan be generated by mutating nucleic acid encoding the mouse variabledomain sequence so that the framework residues thereof are changed toacceptor human framework residues, or by mutating nucleic acid encodingthe human variable domain sequence so that the hypervariable domainresidues are changed to non-human residues, or by synthesizing nucleicacid encoding the desired sequence, etc.

In the examples herein, hypervariable region-grafted variants weregenerated by Kunkel mutagenesis of nucleic acid encoding the humanacceptor sequences, using a separate oligonucleotide for eachhypervariable region. Kunkel et al., Methods Enzymol. 154:367-382(1987). Appropriate changes can be introduced within the frameworkand/or hypervariable region, using routine techniques, to correct andre-establish proper hypervariable region-antigen interactions.

Phage(mid) display (also referred to herein as phage display in somecontexts) can be used as a convenient and fast method for generating andscreening many different potential variant antibodies in a librarygenerated by sequence randomization. However, other methods for makingand screening altered antibodies are available to the skilled person.

Phage(mid) display technology has provided a powerful tool forgenerating and selecting novel proteins which bind to a ligand, such asan antigen. Using the techniques of phage(mid) display allows thegeneration of large libraries of protein variants which can be rapidlysorted for those sequences that bind to a target molecule with highaffinity. Nucleic acids encoding variant polypeptides are generallyfused to a nucleic acid sequence encoding a viral coat protein, such asthe gene III protein or the gene VIII protein. Monovalent phagemiddisplay systems where the nucleic acid sequence encoding the protein orpolypeptide is fused to a nucleic acid sequence encoding a portion ofthe gene III protein have been developed. (Bass, S., Proteins, 8:309(1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology,3:205 (1991)). In a monovalent phagemid display system, the gene fusionis expressed at low levels and wild type gene III proteins are alsoexpressed so that infectivity of the particles is retained. Methods ofgenerating peptide libraries and screening those libraries have beendisclosed in many patents (e.g. U.S. Pat. No. 5,723,286, U.S. Pat. No.5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S.Pat. No. 5,498,530).

Libraries of antibodies or antigen binding polypeptides have beenprepared in a number of ways including by altering a single gene byinserting random DNA sequences or by cloning a family of related genes.Methods for displaying antibodies or antigen binding fragments usingphage(mid) display have been described in U.S. Pat. Nos. 5,750,373,5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727.The library is then screened for expression of antibodies or antigenbinding proteins with the desired characteristics.

Methods of substituting an amino acid of choice into a template nucleicacid are well established in the art, some of which are describedherein. For example, hypervariable region residues can be substitutedusing the Kunkel method. See, e.g., Kunkel et al., Methods Enzymol.154:367-382 (1987).

The sequence of oligonucleotides includes one or more of the designedcodon sets for the hypervariable region residues to be altered. A codonset is a set of different nucleotide triplet sequences used to encodedesired variant amino acids. Codon sets can be represented using symbolsto designate particular nucleotides or equimolar mixtures of nucleotidesas shown in below according to the IUB code.

IUB Codes

G Guanine

A Adenine

T Thymine

C Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S (C or G)

W (A or T)

H (A or C or T)

B (C or G or T)

V (A or C or G)

D (A or G or T)

N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; Vcan be A or G or C; and K can be G or T. This codon set can present 18different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr,Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Oligonucleotide or primer sets can be synthesized using standardmethods. A set of oligonucleotides can be synthesized, for example, bysolid phase synthesis, containing sequences that represent all possiblecombinations of nucleotide triplets provided by the codon set and thatwill encode the desired group of amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art. Such sets of nucleotides havingcertain codon sets can be synthesized using commercial nucleic acidsynthesizers (available from, for example, Applied Biosystems, FosterCity, Calif.), or can be obtained commercially (for example, from LifeTechnologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used according to the invention, have sequencesthat allow for hybridization to a variable domain nucleic acid templateand also can include restriction enzyme sites for cloning purposes.

In one method, nucleic acid sequences encoding variant amino acids canbe created by oligonucleotide-mediated mutagenesis. This technique iswell known in the art as described by Zoller et al. Nucleic Acids Res.10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variantamino acids are created by hybridizing an oligonucleotide set encodingthe desired codon sets to a DNA template, where the template is thesingle-stranded form of the plasmid containing a variable region nucleicacid template sequence. After hybridization, DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will contain thecodon sets as provided by the oligonucleotide set.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation(s). This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Nat'l. Acad. Sci. USA, 75:5765 (1978).

The DNA template is generated by those vectors that are either derivedfrom bacteriophage M13 vectors (the commercially available M13mp18 andM13mp19 vectors are suitable), or those vectors that contain asingle-stranded phage origin of replication as described by Viera etal., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutatedcan be inserted into one of these vectors in order to generatesingle-stranded template. Production of the single-stranded template isdescribed in sections 4.21-4.41 of Sambrook et al., above.

To alter the native DNA sequence, the oligonucleotide is hybridized tothe single stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually T7 DNA polymerase or the Klenowfragment of DNA polymerase I, is then added to synthesize thecomplementary strand of the template using the oligonucleotide as aprimer for synthesis. A heteroduplex molecule is thus formed such thatone strand of DNA encodes the mutated form of gene 1, and the otherstrand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitablehost cell, usually a prokaryote such as E. coli JM101. After growing thecells, they are plated onto agarose plates and screened using theoligonucleotide primer radiolabelled with a 32-Phosphate to identify thebacterial colonies that contain the mutated DNA.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: The singlestranded oligonucleotide is annealed to the single-stranded template asdescribed above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTT), is combined with a modifiedthiodeoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham). This mixture is added to the template-oligonucleotidecomplex. Upon addition of DNA polymerase to this mixture, a strand ofDNA identical to the template except for the mutated bases is generated.In addition, this new strand of DNA will contain dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonucleasedigestion. After the template strand of the double-stranded heteroduplexis nicked with an appropriate restriction enzyme, the template strandcan be digested with ExoIII nuclease or another appropriate nucleasepast the region that contains the site(s) to be mutagenized. Thereaction is then stopped to leave a molecule that is only partiallysingle-stranded. A complete double-stranded DNA homoduplex is thenformed using DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplexmolecule can then be transformed into a suitable host cell.

As indicated previously the sequence of the oligonucleotide set is ofsufficient length to hybridize to the template nucleic acid and mayalso, but does not necessarily, contain restriction sites. The DNAtemplate can be generated by those vectors that are either derived frombacteriophage M13 vectors or vectors that contain a single-strandedphage origin of replication as described by Viera et al. Meth. Enzymol.,153:3 (1987). Thus, the DNA that is to be mutated must be inserted intoone of these vectors in order to generate single-stranded template.Production of the single-stranded template is described in sections4.21-4.41 of Sambrook et al., supra.

According to another method, a library can be generated by providingupstream and downstream oligonucleotide sets, each set having aplurality of oligonucleotides with different sequences, the differentsequences established by the codon sets provided within the sequence ofthe oligonucleotides. The upstream and downstream oligonucleotide sets,along with a variable domain template nucleic acid sequence, can be usedin a polymerase chain reaction to generate a “library” of PCR products.The PCR products can be referred to as “nucleic acid cassettes”, as theycan be fused with other related or unrelated nucleic acid sequences, forexample, viral coat proteins and dimerization domains, using establishedmolecular biology techniques.

The sequence of the PCR primers includes one or more of the designedcodon sets for the solvent accessible and highly diverse positions in ahypervariable region. As described above, a codon set is a set ofdifferent nucleotide triplet sequences used to encode desired variantamino acids.

Antibody selectants that meet the desired criteria, as selected throughappropriate screening/selection steps can be isolated and cloned usingstandard recombinant techniques.

Antibody Fragments

The present invention encompasses antibody fragments. Antibody fragmentsmay be generated by traditional means, such as enzymatic digestion, orby recombinant techniques. In certain circumstances there are advantagesof using antibody fragments, rather than whole antibodies. The smallersize of the fragments allows for rapid clearance, and may lead toimproved access to solid tumors. For a review of certain antibodyfragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody. See, e.g., Sims et al.(1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol.196:901. Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. See, e.g., Carter et al. (1992) Proc.Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol.,151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human Antibodies

Human antibodies of the invention can be constructed by combining Fvclone variable domain sequence(s) selected from human-derived phagedisplay libraries with known human constant domain sequences(s) asdescribed above. Alternatively, human monoclonal antibodies of theinvention can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by HVR grafting, thistechnique provides completely human antibodies, which have no FR or HVRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is for FGF19 andthe other is for any other antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of FGF19. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress FGF19. These antibodies possess a FGF19-binding arm and an armwhich binds a cytotoxic agent, such as, e.g., saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking method. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-dized to form the antibody heterodimers.

This method can also be utilized for the production of antibodyhomodimers. The “diabody” technology described by Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. In certain embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In certain embodiments, amultivalent antibody comprises (or consists of) three to about eightantigen binding sites. In one such embodiment, a multivalent antibodycomprises (or consists of) four antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (for example, twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (for example, four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. (1997) TIBTECH 15:26-32. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

For example, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. Such variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function.

Examples of such antibody variants are described, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which further improve ADCC, forexample, substitutions at positions 298, 333, and/or 334 of the Fcregion (Eu numbering of residues). Such substitutions may occur incombination with any of the variations described above.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for many applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the antibody are measured to ensurethat only the desired properties are maintained. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks FcγR binding(hence likely lacking ADCC activity), but retains FcRn binding ability.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-92 (1991). Non-limiting examples of invitro assays to assess ADCC activity of a molecule of interest isdescribed in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I., et al.Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, for example, Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

Other antibody variants having one or more amino acid substitutions areprovided. Sites of interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions.” More substantial changes, denominated“exemplary substitutions” are provided in Table 1, or as furtherdescribed below in reference to amino acid classes. Amino acidsubstitutions may be introduced into an antibody of interest and theproducts screened, e.g., for a desired activity, such as improvedantigen binding, decreased immunogenicity, improved ADCC or CDC, etc.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Modifications in the biological properties of an antibody may beaccomplished by selecting substitutions that affect (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Amino acids may be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. An exemplary substitutional variant is an affinity maturedantibody, which may be conveniently generated using phage display-basedaffinity maturation techniques. Briefly, several hypervariable regionsites (e.g. 6-7 sites) are mutated to generate all possible amino acidsubstitutions at each site. The antibodies thus generated are displayedfrom filamentous phage particles as fusions to at least part of a phagecoat protein (e.g., the gene III product of M13) packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. binding affinity). In order to identifycandidate hypervariable region sites for modification, scanningmutagenesis (e.g., alanine scanning) can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according totechniques known in the art, including those elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening using techniques known in the art, including those describedherein, and variants with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter, Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

In another aspect, the invention provides antibodies comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

In yet another aspect, it may be desirable to create cysteine engineeredantibodies, e.g., “thioMAbs,” in which one or more residues of anantibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, as described further herein. Incertain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region.

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Activity Assays

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

In one aspect, assays are provided for identifying anti-19 antibodiesthereof having biological activity. Biological activity may include,e.g., the modulation of one or more aspects of FGF19-associated effects,including but not limited to FGF19 binding, FGFR4 activation, FGFR4downstream molecular signaling, disruption of FGFR4 binding to FGF19,FGFR4 multimerization, expression of a CYP7α1 gene, phosphorylation ofFGFR4, MAPK, FRS2 and/or ERK2, activation of β-catenin, FGF19-promotedcell migration, and/or disruption of any biologically relevant FGF19and/or FGFR4 biological pathway, and/or treatment and/or prevention of atumor, cell proliferative disorder or a cancer; and/or treatment orprevention of a disorder associated with FGF19 expression and/oractivity (such as increased FGF19 expression and/or activity).

In certain embodiments, an antibody of the invention is tested for itsability to inhibit, reduce, and/or block FGF19-induced repression ofexpression of a CYP7α1 gene in a cell exposed to FGF19, using methodsknown in the art, e.g., as described in co-owned U.S. patent applicationSer. No. 11/673,411, filed Feb. 9, 2007. In certain embodiments, anantibody of the invention is tested for its ability to inhibit, reduce,and/or block FGF19-induced phosphorylation of FGFR4, MAPK, FRS2 and/orERK2 in a cell exposed to FGF19, using methods known in the art (e.g.,as described in co-owned U.S. patent application Ser. No. 11/673,411,filed Feb. 9, 2007) or exemplified herein. In certain embodiments, anantibody of the invention is tested for its ability to inhibit, reduce,and/or block FGF19-promoted cell (e.g., a tumor cell, e.g., an HCT116cell) migration, using methods known in the art (e.g., as described inco-owned U.S. patent application Ser. No. 11/673,411, filed Feb. 9,2007). In certain embodiments, an antibody of the invention is testedfor its ability to inhibit, reduce, and/or block Wnt pathway activationin a cell. In some embodiments, Wnt pathway activation comprises one ormore of β-catenin immunoreactivity, tyrosine phosphorylation ofβ-catenin, expression of Wnt target genes, β-catenin mutation, andE-cadherin binding to β-catenin. Detection of Wnt pathway activation isknown in the art, and some examples are described and exemplified in,e.g., co-owned U.S. patent application Ser. No. 11/673,411, filed Feb.9, 2007.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc. In some embodiments, an antibody of the invention is tested for itsability to block FGF19 binding to FGFR4, for example as exemplifiedherein. In another aspect, competition assays may be used to identify amonoclonal antibody that competes with any of the anti-FGF19 antibodiesdescribed herein for binding to FGF19. In certain embodiments, such acompeting antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by any of the anti-FGF19antibodies described herein. Exemplary competition assays include, butare not limited to, routine assays such as those provided in Harlow andLane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). Detailed exemplary methods formapping an epitope to which an antibody binds are provided in Morris(1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol.66 (Humana Press, Totowa, N.J.). Two antibodies are said to bind to thesame epitope if each blocks binding of the other by 50% or more.

In an exemplary competition assay, immobilized FGF19 is incubated in asolution comprising a first labeled antibody that binds to FGF19 and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to FGF19. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized FGF19 is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to FGF19,excess unbound antibody is removed, and the amount of label associatedwith immobilized FGF19 is measured. If the amount of label associatedwith immobilized FGF19 is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to FGF19.

The purified immunoglobulins can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the immunoglobulins producedherein are analyzed for their biological activity. In some embodiments,the immunoglobulins of the present invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. An illustrative antigen binding assay isprovided below in the Examples section.

The purified antibodies can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies of the present invention are tested for their antigen bindingactivity. The antigen binding assays that are known in the art and canbe used herein include without limitation any direct or competitivebinding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. Illustrative antigen binding assay are providedbelow in the Examples section.

In some embodiments, the present invention contemplates alteredantibodies that possess some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried outto confirm that the antibody is unable to bind C1q and hence lacks CDCactivity. To assess complement activation, a CDC assay, e.g. asdescribed in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996),may be performed. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art.

In some embodiments, the invention provides altered antibodies thatpossess increased effector functions and/or increased half-life.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a. Generating Antibodies Using Prokaryotic Host Cells:

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM.TM.-11 may be utilized in making arecombinant vector which can be used to transform susceptible host cellssuch as E. coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB−strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coliλ, 1776 (ATCC31,537) and E. coli RV308(ATCC 31,608) are also suitable. These examplesare illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

ii. Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably referredto as “antibody-drug conjugates,” or “ADCs”) comprising an antibodyconjugated to one or more cytotoxic agents, such as a chemotherapeuticagent, a drug, a growth inhibitory agent, a toxin (e.g., a proteintoxin, an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Immunoconjugates have been used for the local delivery of cytotoxicagents, i.e., drugs that kill or inhibit the growth or proliferation ofcells, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion inPharmacology 5:543-549; Wu et al (2005) Nature Biotechnology23(9):1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos(1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer(1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278).Immunoconjugates allow for the targeted delivery of a drug moiety to atumor, and intracellular accumulation therein, where systemicadministration of unconjugated drugs may result in unacceptable levelsof toxicity to normal cells as well as the tumor cells sought to beeliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe(1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological And ClinicalApplications (A. Pinchera et al., eds) pp. 475-506. Both polyclonalantibodies and monoclonal antibodies have been reported as useful inthese strategies (Rowland et al., (1986) Cancer Immunol. Immunother.21:183-87). Drugs used in these methods include daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins usedin antibody-toxin conjugates include bacterial toxins such as diphtheriatoxin, plant toxins such as ricin, small molecule toxins such asgeldanamycin (Mandler et al (2000) J. Nat. Cancer Inst.92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791),maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928;Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins may exerttheir cytotoxic effects by mechanisms including tubulin binding, DNAbinding, or topoisomerase inhibition. Some cytotoxic drugs tend to beinactive or less active when conjugated to large antibodies or proteinreceptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and 111In or 90Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a huCD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody-drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andother cancers. MLN-2704 (Millennium Pharm., BZL Biologics, ImmunogenInc.), an antibody-drug conjugate composed of the anti-prostate specificmembrane antigen (PSMA) monoclonal antibody linked to the maytansinoiddrug moiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnol. 21(7):778-784)and are under therapeutic development.

In certain embodiments, an immunoconjugate comprises an antibody and achemotherapeutic agent or other toxin. Chemotherapeutic agents useful inthe generation of immunoconjugates are described herein (e.g., above).Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×105 HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ1I,α2I, α3I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which is anantifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc99m or I123, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc⁹⁹m or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds expressly contemplate, but are not limited to, ADCprepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). Seepages 467-498, 2003-2004 Applications Handbook and Catalog.

Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC), an antibody (Ab) is conjugated toone or more drug moieties (D), e.g. about 1 to about 20 drug moietiesper antibody, through a linker (L). The ADC of Formula I may be preparedby several routes, employing organic chemistry reactions, conditions,and reagents known to those skilled in the art, including: (1) reactionof a nucleophilic group of an antibody with a bivalent linker reagent,to form Ab-L, via a covalent bond, followed by reaction with a drugmoiety D; and (2) reaction of a nucleophilic group of a drug moiety witha bivalent linker reagent, to form D-L, via a covalent bond, followed byreaction with the nucleophilic group of an antibody. Additional methodsfor preparing ADC are described herein.Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington: The Science and Practice of Pharmacy 20thedition (2000)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

An antibody of the present invention may be used in, for example, invitro, ex vivo and in vivo therapeutic methods.

The invention provides methods and compositions useful for modulatingdisease states associated with expression and/or activity of FGF19and/or FGFR4, such as increased expression and/or activity or undesiredexpression and/or activity, said methods comprising administration of aneffective dose of an anti-FGF19 antibody to an individual in need ofsuch treatment. In some embodiments, the disease state is associatedwith increased expression of FGF19, and the disease state comprisescholestasis or dysregulation of bile acid metabolism.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder, the methodscomprising administering an effective amount of an anti-FGF19 antibodyto an individual in need of such treatment.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder associated withincreased expression and/or activity of FGF19, the methods comprisingadministering an effective amount of an anti-FGF19 antibody to anindividual in need of such treatment.

In one aspect, the invention provides methods for treating or preventinga tumor, a cancer, and/or a cell proliferative disorder associated withincreased expression and/or activity of FGFR4, the methods comprisingadministering an effective amount of an anti-FGF19 antibody to anindividual in need of such treatment.

In one aspect, the invention provides methods for treating and/orpreventing a liver disorder, the methods comprising administering aneffective amount of an anti-FGF19 antibody to an individual in need ofsuch treatment. In some embodiments, the liver disorder is cirrhosis.

In one aspect, the invention provides methods for treating and/orpreventing a wasting disorder, the methods comprising administering aneffective amount of an anti-FGF19 antibody to an individual in need ofsuch treatment. In some embodiments, the individual has a tumor, acancer, and/or a cell proliferative disorder.

It is understood that any suitable anti-FGF19 antibody may be used inmethods of treatment, including monoclonal and/or polyclonal antibodies,a human antibody, a chimeric antibody, an affinity-matured antibody, ahumanized antibody, and/or an antibody fragment. In some embodiments,any anti-FGF19 antibody described herein is used for treatment.

Moreover, at least some of the antibodies of the invention can bindantigen from other species. Accordingly, the antibodies of the inventioncan be used to bind specific antigen activity, e.g., in a cell culturecontaining the antigen, in human subjects or in other mammalian subjectshaving the antigen with which an antibody of the invention cross-reacts(e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig ormouse). In one embodiment, the antibody of the invention can be used forinhibiting antigen activities by contacting the antibody with theantigen such that antigen activity is inhibited. Preferably, the antigenis a human protein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor binding an antigen in an individual suffering from a disorderassociated with increased antigen expression and/or activity, comprisingadministering to the subject an antibody of the invention such that theantigen in the subject is bound. Preferably, the antigen is a humanprotein molecule and the subject is a human subject. Alternatively, thesubject can be a mammal expressing the antigen with which an antibody ofthe invention binds. Still further the subject can be a mammal intowhich the antigen has been introduced (e.g., by administration of theantigen or by expression of an antigen transgene). An antibody of theinvention can be administered to a human subject for therapeuticpurposes. Moreover, an antibody of the invention can be administered toa non-human mammal expressing an antigen with which the immunoglobulincross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration).

The antibodies of the invention can be used to treat, inhibit, delayprogression of, prevent/delay recurrence of, ameliorate, or preventdiseases, disorders or conditions associated with expression and/oractivity of one or more antigen molecules.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with one or more cytotoxic agent(s) is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thetarget cell to which it binds. In one embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the target cell. In oneembodiment, the cytotoxic agent targets or interferes with microtubulepolymerization. Examples of such cytotoxic agents include any of thechemotherapeutic agents noted herein (such as a maytansinoid,auristatin, dolastatin, or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

In any of the methods herein, one may administer to the subject orpatient along with the antibody herein an effective amount of a secondmedicament (where the antibody herein is a first medicament), which isanother active agent that can treat the condition in the subject thatrequires treatment. For instance, an antibody of the invention may beco-administered with another antibody, chemotherapeutic agent(s)(including cocktails of chemotherapeutic agents), anti-angiogenicagent(s), immunosuppressive agents(s), cytokine(s), cytokineantagonist(s), and/or growth-inhibitory agent(s). The type of suchsecond medicament depends on various factors, including the type ofdisorder, such as cancer or an autoimmune disorder, the severity of thedisease, the condition and age of the patient, the type and dose offirst medicament employed, etc.

Where an antibody of the invention inhibits tumor growth, for example,it may be particularly desirable to combine it with one or more othertherapeutic agents that also inhibit tumor growth. For instance, anantibody of the invention may be combined with an anti-angiogenic agent,such as an anti-VEGF antibody (e.g., AVASTIN®) and/or anti-ErbBantibodies (e.g. HERCEPTIN® trastuzumab anti-HER2 antibody or ananti-HER2 antibody that binds to Domain II of HER2, such as OMNITARG™pertuzumab anti-HER2 antibody) in a treatment scheme, e.g. in treatingany of the disease described herein, including colorectal cancer, lungcancer, hepatocellular carcinoma, breast cancer and/or pancreaticcancer. Alternatively, or additionally, the patient may receive combinedradiation therapy (e.g. external beam irradiation or therapy with aradioactive labeled agent, such as an antibody). Such combined therapiesnoted above include combined administration (where the two or moreagents are included in the same or separate formulations), and separateadministration, in which case, administration of the antibody of theinvention can occur prior to, and/or following, administration of theadjunct therapy or therapies. In addition, combining an antibody of thisinvention with a relatively non-cytotoxic agent such as another biologicmolecule, e.g., another antibody is expected to reduce cytotoxicityversus combining the antibody with a chemotherapeutic agent of otheragent that is highly toxic to cells.

Treatment with a combination of the antibody herein with one or moresecond medicaments preferably results in an improvement in the signs orsymptoms of cancer. For instance, such therapy may result in animprovement in survival (overall survival and/or progression-freesurvival) relative to a patient treated with the second medicament only(e.g., a chemotherapeutic agent only), and/or may result in an objectiveresponse *(partial or complete, preferably complete). Moreover,treatment with the combination of an antibody herein and one or moresecond medicament(s) preferably results in an additive, and morepreferably synergistic (or greater than additive), therapeutic benefitto the patient. Preferably, in this combination method the timingbetween at least one administration of the second medicament and atleast one administration of the antibody herein is about one month orless, more preferably, about two weeks or less.

For treatment of cancers, the second medicament is preferably anotherantibody, chemotherapeutic agent (including cocktails ofchemotherapeutic agents), anti-angiogenic agent, immunosuppressiveagent, prodrug, cytokine, cytokine antagonist, cytotoxic radiotherapy,corticosteroid, anti-emetic, cancer vaccine, analgesic, anti-vascularagent, and/or growth-inhibitory agent. The cytotoxic agent includes anagent interacting with DNA, the antimetabolites, the topoisomerase I orII inhibitors, or the spindle inhibitor or stabilizer agents (e.g.,preferably vinca alkaloid, more preferably selected from vinblastine,deoxyvinblastine, vincristine, vindesine, vinorelbine, vinepidine,vinfosiltine, vinzolidine and vinfunine), or any agent used inchemotherapy such as 5-FU, a taxane, doxorubicin, or dexamethasone.

In another embodiment, the second medicament is another antibody used totreat cancers such as those directed against the extracellular domain ofthe HER2/neu receptor, e.g., trastuzumab, or one of its functionalfragments, pan-HER inhibitor, a Src inhibitor, a MEK inhibitor, or anEGFR inhibitor (e.g., an anti-EGFR antibody (such as one inhibiting thetyrosine kinase activity of the EGFR), which is preferably the mousemonoclonal antibody 225, its mouse-man chimeric derivative C225, or ahumanized antibody derived from this antibody 225 or derived naturalagents, dianilinophthalimides, pyrazolo- or pyrrolopyridopyrimidines,quinazilines, gefitinib, erlotinib, cetuximab, ABX-EFG, canertinib,EKB-569 and PKI-166), or dual-EGFR/HER-2 inhibitor such as lapatanib.Additional second medicaments include alemtuzumab (CAMPATH™), FavID(IDKLH), CD20 antibodies with altered glycosylation, such asGA-101/GLYCART™, oblimersen (GENASENSE™), thalidomide and analogsthereof, such as lenalidomide (REVLIMID™), imatinib, sorafenib,ofatumumab (HUMAX-CD20™), anti-CD40 antibody, e.g. SGN-40, andanti-CD-80 antibody, e.g. galiximab.

The anti-emetic agent is preferably ondansetron hydrochloride,granisetron hydrochloride, metroclopramide, domperidone, haloperidol,cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine,or tropisetron. The vaccine is preferably GM-CSF DNA and cell-basedvaccines, dendritic cell vaccine, recombinant viral vaccines, heat shockprotein (HSP) vaccines, allogeneic or autologous tumor vaccines. Theanalgesic agent preferably is ibuprofen, naproxen, choline magnesiumtrisalicylate, or oxycodone hydrochloride. The anti-vascular agentpreferably is bevacizumab, or rhuMAb-VEGF. Further second medicamentsinclude anti-proliferative agents such a farnesyl protein transferaseinhibitors, anti-VEGF inhibitors, p53 inhibitors, or PDGFR inhibitors.The second medicament herein includes also biologic-targeted therapysuch as treatment with antibodies as well as small-molecule-targetedtherapy, for example, against certain receptors.

Many anti-angiogenic agents have been identified and are known in theart, including those listed herein, e.g., listed under Definitions, andby, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al.,Nature Reviews: Drug Discovery, 3:391-400 (2004); and Sato Int. J. Clin.Oncol., 8:200-206 (2003). See also, US Patent Application US20030055006.In one embodiment, an anti-FGF19 antibody is used in combination with ananti-VEGF neutralizing antibody (or fragment) and/or another VEGFantagonist or a VEGF receptor antagonist including, but not limited to,for example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3,neuropillins (e.g., NRP1, NRP2)) fragments, aptamers capable of blockingVEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weightinhibitors of VEGFR tyrosine kinases (RTK), antisense strategies forVEGF, ribozymes against VEGF or VEGF receptors, antagonist variants ofVEGF; and any combinations thereof. Alternatively, or additionally, twoor more angiogenesis inhibitors may optionally be co-administered to thepatient in addition to VEGF antagonist and other agent. In certainembodiment, one or more additional therapeutic agents, e.g., anti-canceragents, can be administered in combination with anti-FGF19 antibody, theVEGF antagonist, and an anti-angiogenesis agent.

Chemotherapeutic agents useful herein are described supra, e.g., in thedefinition of “chemotherapeutic agent”.

Exemplary second medicaments include an alkylating agent, a folateantagonist, a pyrimidine antagonist, a cytotoxic antibiotic, a platinumcompound or platinum-based compound, a taxane, a vinca alkaloid, a c-Kitinhibitor, a topoisomerase inhibitor, an anti-angiogenesis inhibitorsuch as an anti-VEGF inhibitor, a HER-2 inhibitor, an EGFR inhibitor ordual EGFR/HER-2 kinase inhibitor, an anti-estrogen such as fulvestrant,and a hormonal therapy agent, such as carboplatin, cisplatin,gemcitabine, capecitabine, epirubicin, tamoxifen, an aromataseinhibitor, and prednisone. Most preferably, the cancer is colorectalcancer and the second medicament is an EGFR inhibitor such as erlotinib,an anti-VEGF inhibitor such as bevacizumab, or is cetuximab, arinotecan,irinotecan, or FOLFOX, or the cancer is breast cancer an the secondmedicament is an anti-estrogen modulator such as fulvestrant, tamoxifenor an aromatase inhibitor such as letrozole, exemestane, or anastrozole,or is a VEGF inhibitor such as bevacizumab, or is a chemotherapeuticagent such as doxorubicin, and/or a taxane such as paclitaxel, or is ananti-HER-2 inhibitor such as trastuzumab, or a dual EGFR/HER-2 kinaseinhibitor such as lapatinib or a HER-2 downregulator such as 17AAG(geldanamycin derivative that is a heat shock protein [Hsp] 90 poison)(for example, for breast cancers that have progressed on trastuzumab).In other embodiments, the cancer is lung cancer, such as small-cell lungcancer, and the second medicament is a VEGF inhibitor such asbevacizumab, or an EGFR inhibitor such as, e.g., erlotinib or a c-Kitinhibitor such as e.g., imatinib. In other embodiments, the cancer isliver cancer, such as hepatocellular carcinoma, and the secondmedicament is an EGFR inhibitor such as erlotinib, a chemotherapeuticagent such as doxorubicin or irinotecan, a taxane such as paclitaxel,thalidomide and/or interferon. Further, a preferred chemotherapeuticagent for front-line therapy of cancer is taxotere, alone in combinationwith other second medicaments. Most preferably, if chemotherapy isadministered, it is given first, followed by the antibodies herein.

Such second medicaments may be administered within 48 hours after theantibodies herein are administered, or within 24 hours, or within 12hours, or within 3-12 hours after said agent, or may be administeredover a pre-selected period of time, which is preferably about 1 to 2days. Further, the dose of such agent may be sub-therapeutic.

The antibodies herein can be administered concurrently, sequentially, oralternating with the second medicament or upon non-responsiveness withother therapy. Thus, the combined administration of a second medicamentincludes co-administration (concurrent administration), using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) medicaments simultaneously exert theirbiological activities. All these second medicaments may be used incombination with each other or by themselves with the first medicament,so that the express “second medicament” as used herein does not mean itis the only medicament besides the first medicament, respectively. Thus,the second medicament need not be one medicament, but may constitute orcomprise more than one such drug.

These second medicaments as set forth herein are generally used in thesame dosages and with administration routes as the first medicaments, orabout from 1 to 99% of the dosages of the first medicaments. If suchsecond medicaments are used at all, preferably, they are used in loweramounts than if the first medicament were not present, especially insubsequent dosings beyond the initial dosing with the first medicament,so as to eliminate or reduce side effects caused thereby.

The invention also provides methods and compositions for inhibiting orpreventing relapse tumor growth or relapse cancer cell growth. Relapsetumor growth or relapse cancer cell growth is used to describe acondition in which patients undergoing or treated with one or morecurrently available therapies (e.g., cancer therapies, such aschemotherapy, radiation therapy, surgery, hormonal therapy and/orbiological therapy/immunotherapy, anti-VEGF antibody therapy,particularly a standard therapeutic regimen for the particular cancer)is not clinically adequate to treat the patients or the patients are nolonger receiving any beneficial effect from the therapy such that thesepatients need additional effective therapy. As used herein, the phrasecan also refer to a condition of the “non-responsive/refractory”patient, e.g., which describe patients who respond to therapy yet sufferfrom side effects, develop resistance, do not respond to the therapy, donot respond satisfactorily to the therapy, etc. In various embodiments,a cancer is relapse tumor growth or relapse cancer cell growth where thenumber of cancer cells has not been significantly reduced, or hasincreased, or tumor size has not been significantly reduced, or hasincreased, or fails any further reduction in size or in number of cancercells. The determination of whether the cancer cells are relapse tumorgrowth or relapse cancer cell growth can be made either in vivo or invitro by any method known in the art for assaying the effectiveness oftreatment on cancer cells, using the art-accepted meanings of “relapse”or “refractory” or “non-responsive” in such a context. A tumor resistantto anti-VEGF treatment is an example of a relapse tumor growth.

The invention provides methods of blocking or reducing relapse tumorgrowth or relapse cancer cell growth in a subject by administering oneor more anti-FGF19 antibody to block or reduce the relapse tumor growthor relapse cancer cell growth in subject. In certain embodiments, theantagonist can be administered subsequent to the cancer therapeutic. Incertain embodiments, the anti-FGF19 antibody is administeredsimultaneously with cancer therapy. Alternatively, or additionally, theanti-FGF19 antibody therapy alternates with another cancer therapy,which can be performed in any order. The invention also encompassesmethods for administering one or more inhibitory antibodies to preventthe onset or recurrence of cancer in patients predisposed to havingcancer. Generally, the subject was or is concurrently undergoing cancertherapy. In one embodiment, the cancer therapy is treatment with ananti-angiogenesis agent, e.g., a VEGF antagonist. The anti-angiogenesisagent includes those known in the art and those found under theDefinitions herein. In one embodiment, the anti-angiogenesis agent is ananti-VEGF neutralizing antibody or fragment (e.g., humanized A4.6.1,AVASTIN® (Genentech, South San Francisco, Calif.), Y0317, M4, G6, B20,2C3, etc.). See, e.g., U.S. Pat. Nos. 6,582,959, 6,884,879, 6,703,020;WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1; US PatentApplications 20030206899, 20030190317, 20030203409, and 20050112126;Popkov et al., Journal of Immunological Methods 288:149-164 (2004); and,WO2005012359. Additional agents can be administered in combination withVEGF antagonist and an anti-FGF19 antibody for blocking or reducingrelapse tumor growth or relapse cancer cell growth.

The antibodies of the invention (and adjunct therapeutic agent) is/areadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibodies are suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, e.g. by injections, suchas intravenous or subcutaneous injections, depending in part on whetherthe administration is brief or chronic.

The location of the binding target of an antibody of the invention maybe taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described, e.g., in Marasco, Gene Therapy 4: 11-15(1997); Kontermann, Methods 34: 163-170 (2004); U.S. Pat. Nos. 6,004,940and 6,329,173; U.S. Patent Application Publication No. 2003/0104402, andPCT Publication No. WO2003/077945. See also, for example, WO96/07321published Mar. 14, 1996, concerning the use of gene therapy to generateintracellular antibodies.

Intracellular expression of an intrabody may be effected by introducinga nucleic acid encoding the desired antibody or antigen-binding portionthereof (lacking the wild-type leader sequence and secretory signalsnormally associated with the gene encoding that antibody orantigen-binding fragment) into a target cell. One or more nucleic acidsencoding all or a portion of an antibody of the invention can bedelivered to a target cell, such that one or more intrabodies areexpressed which are capable of binding to an intracellular targetpolypeptide and modulating the activity of the target polypeptide. Anystandard method of introducing nucleic acids into a cell may be used,including, but not limited to, microinjection, ballistic injection,electroporation, calcium phosphate precipitation, liposomes, andtransfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest.

In certain embodiments, nucleic acid (optionally contained in a vector)may be introduced into a patient's cells by in vivo and ex vivo methods.In one example of in vivo delivery, nucleic acid is injected directlyinto the patient, e.g., at the site where therapeutic intervention isrequired. In a further example of in vivo delivery, nucleic acid isintroduced into a cell using transfection with viral vectors (such asadenovirus, Herpes simplex I virus, or adeno-associated virus) andlipid-based systems (useful lipids for lipid-mediated transfer of thegene are DOTMA, DOPE and DC-Chol, for example). For review of certaingene marking and gene therapy protocols, see Anderson et al., Science256:808-813 (1992), and WO 93/25673 and the references cited therein. Inan example of ex vivo treatment, a patient's cells are removed, nucleicacid is introduced into those isolated cells, and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). A commonlyused vector for ex vivo delivery of a nucleic acid is a retroviralvector.

In another embodiment, internalizing antibodies are provided. Antibodiescan possess certain characteristics that enhance delivery of antibodiesinto cells, or can be modified to possess such characteristics.Techniques for achieving this are known in the art. For example,cationization of an antibody is known to facilitate its uptake intocells (see, e.g., U.S. Pat. No. 6,703,019). Lipofections or liposomescan also be used to deliver the antibody into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the target protein may be advantageous. For example, based uponthe variable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993).

Entry of antibodies into target cells can be enhanced by other methodsknown in the art. For example, certain sequences, such as those derivedfrom HIV Tat or the Antennapedia homeodomain protein are able to directefficient uptake of heterologous proteins across cell membranes. See,e.g., Chen et al., Proc. Natl. Acad. Sci. USA (1999), 96:4325-4329.

When the binding target of an antibody is located in the brain, certainembodiments of the invention provide for the antibody to traverse theblood-brain barrier. Several art-known approaches exist for transportingmolecules across the blood-brain barrier, including, but not limited to,physical methods, lipid-based methods, stem cell-based methods, andreceptor and channel-based methods.

Physical methods of transporting an antibody across the blood-brainbarrier include, but are not limited to, circumventing the blood-brainbarrier entirely, or by creating openings in the blood-brain barrier.Circumvention methods include, but are not limited to, direct injectioninto the brain (see, e.g., Papanastassiou et al., Gene Therapy 9:398-406 (2002)), interstitial infusion/convection-enhanced delivery(see, e.g., Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080(1994)), and implanting a delivery device in the brain (see, e.g., Gillet al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers™, GuildfordPharmaceutical). Methods of creating openings in the barrier include,but are not limited to, ultrasound (see, e.g., U.S. Patent PublicationNo. 2002/0038086), osmotic pressure (e.g., by administration ofhypertonic mannitol (Neuwelt, E. A., Implication of the Blood-BrainBarrier and its Manipulation, Vols 1 & 2, Plenum Press, N.Y. (1989)),permeabilization by, e.g., bradykinin or permeabilizer A-7 (see, e.g.,U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416), andtransfection of neurons that straddle the blood-brain barrier withvectors containing genes encoding the antibody (see, e.g., U.S. PatentPublication No. 2003/0083299).

Lipid-based methods of transporting an antibody across the blood-brainbarrier include, but are not limited to, encapsulating the antibody inliposomes that are coupled to antibody binding fragments that bind toreceptors on the vascular endothelium of the blood-brain barrier (see,e.g., U.S. Patent Application Publication No. 20020025313), and coatingthe antibody in low-density lipoprotein particles (see, e.g., U.S.Patent Application Publication No. 20040204354) or apolipoprotein E(see, e.g., U.S. Patent Application Publication No. 20040131692).

Stem-cell based methods of transporting an antibody across theblood-brain barrier entail genetically engineering neural progenitorcells (NPCs) to express the antibody of interest and then implanting thestem cells into the brain of the individual to be treated. See Behrstocket al. (2005) Gene Ther. 15 Dec. 2005 advanced online publication(reporting that NPCs genetically engineered to express the neurotrophicfactor GDNF reduced symptoms of Parkinson disease when implanted intothe brains of rodent and primate models).

Receptor and channel-based methods of transporting an antibody acrossthe blood-brain barrier include, but are not limited to, usingglucocorticoid blockers to increase permeability of the blood-brainbarrier (see, e.g., U.S. Patent Application Publication Nos.2002/0065259, 2003/0162695, and 2005/0124533); activating potassiumchannels (see, e.g., U.S. Patent Application Publication No.2005/0089473), inhibiting ABC drug transporters (see, e.g., U.S. PatentApplication Publication No. 2003/0073713); coating antibodies with atransferrin and modulating activity of the one or more transferrinreceptors (see, e.g., U.S. Patent Application Publication No.2003/0129186), and cationizing the antibodies (see, e.g., U.S. Pat. No.5,004,697).

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

Diagnostic Methods and Methods of Detection

The anti-FGF19 antibodies of the invention are useful in assaysdetecting FGF19 expression (such as diagnostic or prognostic assays) inspecific cells or tissues wherein the antibodies are labeled asdescribed below and/or are immobilized on an insoluble matrix. However,it is understood that any suitable anti-FGF19 antibody may be used inembodiments involving detection and diagnosis. Some methods for makinganti-FGF19 antibodies are described herein and methods for makinganti-FGF19 antibodies are well known in the art.

In another aspect, the invention provides methods for detection ofFGF19, the methods comprising detecting FGF19-anti-FGF19 antibodycomplex in the sample. The term “detection” as used herein includesqualitative and/or quantitative detection (measuring levels) with orwithout reference to a control.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGF19 expression and/or activity, the methodscomprising detecting FGF19-anti-FGF19 antibody complex in a biologicalsample from an individual having or suspected of having the disorder. Insome embodiments, the FGF19 expression is increased expression orabnormal (undesired) expression.

In another aspect, the invention provides any of the anti-FGF19antibodies described herein, wherein the anti-FGF19 antibody comprises adetectable label.

In another aspect, the invention provides a complex of any of theanti-FGF19 antibodies described herein and FGF19. In some embodiments,the complex is in vivo or in vitro. In some embodiments, the complexcomprises a cancer cell. In some embodiments, the anti-FGF19 antibody isdetectably labeled.

Anti-FGF19 antibodies (e.g., any of the FGF19 antibodies describedherein) can be used for the detection of FGF19 in any one of a number ofwell known detection assay methods.

In one aspect, the invention provides methods for detecting a disorderassociated with FGF19 expression and/or activity, the methods comprisingdetecting FGF19 in a biological sample from an individual. In someembodiments, the FGF19 expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder, such as colorectal cancer, lung cancer,hepatocellular carcinoma, breast cancer and/or pancreatic cancer. Insome embodiment, the biological sample is serum or of a tumor.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGF19expression in an individual's biological sample, if any; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGF19 expression detected instep (a). In some embodiments, increased FGF19 expression in theindividual's biological sample relative to a reference value or controlsample is detected. In some embodiments, decreased FGF19 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected in the individual. In some embodiments, FGF19expression is detected and treatment with an anti-FGF19 antibody isselected. Methods of treating a disorder with an anti-FGF19 antibody aredescribed herein and some methods are exemplified herein.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-FGF19 antibody, furtherwherein FGF19 expression and/or FGFR4 is detected in cells and/or tissuefrom the human patient before, during or after administration of ananti-FGF19 antibody. In some embodiments, FGF19 over-expression isdetected before, during and/or after administration of an anti-FGF19antibody. In some embodiments, FGFR4 expression is detected before,during and/or after administration of an anti-FGF19 antibody. Expressionmay be detected before; during; after; before and during; before andafter; during and after; or before, during and after administration ofan anti-FGF19 antibody. Methods of treating a disorder with ananti-FGF19 antibody are described herein and some methods areexemplified herein.

For example, a biological sample may be assayed for FGF19 by obtainingthe sample from a desired source, admixing the sample with anti-FGF19antibody to allow the antibody to form antibody/FGF19 complex with anyFGF19 present in the mixture, and detecting any antibody/FGF19 complexpresent in the mixture. The biological sample may be prepared for assayby methods known in the art which are suitable for the particularsample. The methods of admixing the sample with antibodies and themethods of detecting antibody/FGF19 complex are chosen according to thetype of assay used. Such assays include immunohistochemistry,competitive and sandwich assays, and steric inhibition assays. Forsample preparation, a tissue or cell sample from a mammal (typically ahuman patient) may be used. Examples of samples include, but are notlimited to, cancer cells such as colon, breast, prostate, ovary, lung,stomach, pancreas, lymphoma, and leukemia cancer cells. FGF19 may alsobe measured in serum. The sample can be obtained by a variety ofprocedures known in the art including, but not limited to surgicalexcision, aspiration or biopsy. The tissue may be fresh or frozen. Inone embodiment, the sample is fixed and embedded in paraffin or thelike. The tissue sample may be fixed (i.e. preserved) by conventionalmethodology (See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology,” 3^(rd) edition (1960) Lee G. Luna,H T (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, NewYork; The Armed Forces Institute of Pathology Advanced LaboratoryMethods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, ArmedForces Institute of Pathology, American Registry of Pathology,Washington, D.C.). One of ordinary skill in the art will appreciate thatthe choice of a fixative is determined by the purpose for which thesample is to be histologically stained or otherwise analyzed. One ofordinary skill in the art will also appreciate that the length offixation depends upon the size of the tissue sample and the fixativeused. By way of example, neutral buffered formalin, Bouin's orparaformaldehyde, may be used to fix a sample. Generally, the sample isfirst fixed and is then dehydrated through an ascending series ofalcohols, infiltrated and embedded with paraffin or other sectioningmedia so that the tissue sample may be sectioned. Alternatively, one maysection the tissue and fix the sections obtained. By way of example, thetissue sample may be embedded and processed in paraffin by conventionalmethodology (See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology”, supra). Examples of paraffin thatmay be used include, but are not limited to, Paraplast, Broloid, andTissuemay. Once the tissue sample is embedded, the sample may besectioned by a microtome or the like (See e.g., “Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology”, supra). Byway of example for this procedure, sections may range from about threemicrons to about five microns in thickness. Once sectioned, the sectionsmay be attached to slides by several standard methods. Examples of slideadhesives include, but are not limited to, silane, gelatin,poly-L-lysine and the like. By way of example, the paraffin embeddedsections may be attached to positively charged slides and/or slidescoated with poly-L-lysine. If paraffin has been used as the embeddingmaterial, the tissue sections are generally deparaffinized andrehydrated to water. The tissue sections may be deparaffinized byseveral conventional standard methodologies. For example, xylenes and agradually descending series of alcohols may be used (See e.g., “Manualof Histological Staining Method of the Armed Forces Institute ofPathology”, supra). Alternatively, commercially availabledeparaffinizing non-organic agents such as Hemo-De7 (CMS, Houston, Tex.)may be used.

Analytical methods for FGF19 all use one or more of the followingreagents: labeled FGF19 analogue, immobilized FGF19 analogue, labeledanti-FGF19 antibody, immobilized anti-FGF19 antibody and stericconjugates. The labeled reagents also are known as “tracers.”

The label used is any detectable functionality that does not interferewith the binding of FGF19 and anti-FGF19 antibody. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected.

The label used is any detectable functionality that does not interferewith the binding of FGF19 and anti-FGF19 antibody. Numerous labels areknown for use in immunoassay, examples including moieties that may bedetected directly, such as fluorochrome, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. Examples of such labels includethe radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David etal., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol.Methods, 40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem.,30: 407-412 (1982). Preferred labels herein are enzymes such ashorseradish peroxidase and alkaline phosphatase. The conjugation of suchlabel, including the enzymes, to the antibody is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., “Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y., 1981), pp. 147-166.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the anti-FGF19 antibody from any FGF19that remains free in solution. This conventionally is accomplished byeither insolubilizing the anti-FGF19 antibody or FGF19 analogue beforethe assay procedure, as by adsorption to a water-insoluble matrix orsurface (Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling(for example, using glutaraldehyde cross-linking), or by insolubilizingthe anti-FGF19 antibody or FGF19 analogue afterward, e.g., byimmunoprecipitation.

The expression of proteins in a sample may be examined usingimmunohistochemistry and staining protocols. Immunohistochemicalstaining of tissue sections has been shown to be a reliable method ofassessing or detecting presence of proteins in a sample.Immunohistochemistry (“IHC”) techniques utilize an antibody to probe andvisualize cellular antigens in situ, generally by chromogenic orfluorescent methods. For sample preparation, a tissue or cell samplefrom a mammal (typically a human patient) may be used. The sample can beobtained by a variety of procedures known in the art including, but notlimited to surgical excision, aspiration or biopsy. The tissue may befresh or frozen. In one embodiment, the sample is fixed and embedded inparaffin or the like. The tissue sample may be fixed (i.e. preserved) byconventional methodology. One of ordinary skill in the art willappreciate that the choice of a fixative is determined by the purposefor which the sample is to be histologically stained or otherwiseanalyzed. One of ordinary skill in the art will also appreciate that thelength of fixation depends upon the size of the tissue sample and thefixative used.

IHC may be performed in combination with additional techniques such asmorphological staining and/or fluorescence in-situ hybridization. Twogeneral methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigen(e.g., FGF19) is determined directly. This direct assay uses a labeledreagent, such as a fluorescent tag or an enzyme-labeled primaryantibody, which can be visualized without further antibody interaction.In a typical indirect assay, unconjugated primary antibody binds to theantigen and then a labeled secondary antibody binds to the primaryantibody. Where the secondary antibody is conjugated to an enzymaticlabel, a chromogenic or fluorogenic substrate is added to providevisualization of the antigen. Signal amplification occurs becauseseveral secondary antibodies may react with different epitopes on theprimary antibody.

The primary and/or secondary antibody used for immunohistochemistrytypically will be labeled with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:

Aside from the sample preparation procedures discussed above, furthertreatment of the tissue section prior to, during or following IHC may bedesired, For example, epitope retrieval methods, such as heating thetissue sample in citrate buffer may be carried out (see, e.g., Leong etal. Appl. Immunohistochem. 4(3):201 (1996)).

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the target proteinantigen in the tissue sample. Appropriate conditions for achieving thiscan be determined by routine experimentation. The extent of binding ofantibody to the sample is determined by using any one of the detectablelabels discussed above. Preferably, the label is an enzymatic label(e.g. HRPO) which catalyzes a chemical alteration of the chromogenicsubstrate such as 3,3′-diaminobenzidine chromogen. Preferably theenzymatic label is conjugated to antibody which binds specifically tothe primary antibody (e.g. the primary antibody is rabbit polyclonalantibody and secondary antibody is goat anti-rabbit antibody).

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g. using a microscope, and stainingintensity criteria, routinely used in the art, may be employed.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer FGF19 analogue tocompete with the test sample FGF19 for a limited number of anti-FGF19antibody antigen-binding sites. The anti-FGF19 antibody generally isinsolubilized before or after the competition and then the tracer andFGF19 bound to the anti-FGF19 antibody are separated from the unboundtracer and FGF19. This separation is accomplished by decanting (wherethe binding partner was preinsolubilized) or by centrifuging (where thebinding partner was precipitated after the competitive reaction). Theamount of test sample FGF19 is inversely proportional to the amount ofbound tracer as measured by the amount of marker substance.Dose-response curves with known amounts of FGF19 are prepared andcompared with the test results to quantitatively determine the amount ofFGF19 present in the test sample. These assays are called ELISA systemswhen enzymes are used as the detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theFGF19 is prepared and used such that when anti-FGF19 antibody binds tothe FGF19 the presence of the anti-FGF19 antibody modifies the enzymeactivity. In this case, the FGF19 or its immunologically activefragments are conjugated with a bifunctional organic bridge to an enzymesuch as peroxidase. Conjugates are selected for use with anti-FGF19antibody so that binding of the anti-FGF19 antibody inhibits orpotentiates the enzyme activity of the label. This method per se iswidely practiced under the name of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small FGF19 fragment so that antibodyto hapten is substantially unable to bind the conjugate at the same timeas anti-FGF19 antibody. Under this assay procedure the FGF19 present inthe test sample will bind anti-FGF19 antibody, thereby allowinganti-hapten to bind the conjugate, resulting in a change in thecharacter of the conjugate hapten, e.g., a change in fluorescence whenthe hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of FGF19or anti-FGF19 antibodies. In sequential sandwich assays an immobilizedanti-FGF19 antibody is used to adsorb test sample FGF19, the test sampleis removed as by washing, the bound FGF19 is used to adsorb a second,labeled anti-FGF19 antibody and bound material is then separated fromresidual tracer. The amount of bound tracer is directly proportional totest sample FGF19. In “simultaneous” sandwich assays the test sample isnot separated before adding the labeled anti-FGF19. A sequentialsandwich assay using an anti-FGF19 monoclonal antibody as one antibodyand a polyclonal anti-FGF19 antibody as the other is useful in testingsamples for FGF19.

The foregoing are merely exemplary detection assays for FGF19. Othermethods now or hereafter developed that use anti-FGF19 antibody for thedetermination of FGF19 are included within the scope hereof, includingthe bioassays described herein.

In one aspect, the invention provides methods to detect (e.g., presenceor absence of or amount) a polynucleotide(s) (e.g., FGF19polynucleotides) in a biological sample from an individual, such as ahuman subject. A variety of methods for detecting polynucleotides can beemployed and include, for example, RT-PCR, taqman, amplificationmethods, polynucleotide microarray, and the like.

Methods for the detection of polynucleotides (such as mRNA) are wellknown and include, for example, hybridization assays using complementaryDNA probes (such as in situ hybridization using labeled FGF19riboprobes), Northern blot and related techniques, and various nucleicacid amplification assays (such as RT-PCR using complementary primersspecific for FGF19, and other amplification type detection methods, suchas, for example, branched DNA, SPIA, Ribo-SPIA, SISBA, TMA and thelike).

Biological samples from mammals can be conveniently assayed for, e.g.,FGF19 mRNAs using Northern, dot blot or PCR analysis. For example,RT-PCR assays such as quantitative PCR assays are well known in the art.In an illustrative embodiment of the invention, a method for detectingFGF19 mRNA in a biological sample comprises producing cDNA from thesample by reverse transcription using at least one primer; amplifyingthe cDNA so produced using an FGF19 polynucleotide as sense andantisense primers to amplify FGF19 cDNAs therein; and detecting thepresence or absence of the amplified FGF19 cDNA. In addition, suchmethods can include one or more steps that allow one to determine theamount (levels) of FGF19 mRNA in a biological sample (e.g. bysimultaneously examining the levels a comparative control mRNA sequenceof a housekeeping gene such as an actin family member). Optionally, thesequence of the amplified FGF19 cDNA can be determined.

Probes and/or primers may be labeled with a detectable marker, such as,for example, a radioisotope, fluorescent compound, bioluminescentcompound, a chemiluminescent compound, metal chelator or enzyme. Suchprobes and primers can be used to detect the presence of FGF 19polynucleotides in a sample and as a means for detecting a cellexpressing FGF19 proteins. As will be understood by the skilled artisan,a great many different primers and probes may be prepared (e.g., basedon the sequences provided in herein) and used effectively to amplify,clone and/or determine the presence or absence of and/or amount of FGF19mRNAs.

Optional methods of the invention include protocols comprising detectionof polynucleotides, such as FGF19 polynucleotide, in a tissue or cellsample using microarray technologies. For example, using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. Theprobes are then hybridized to an array of nucleic acids immobilized on asolid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes that have potential to be expressed in certain disease statesmay be arrayed on a solid support. Hybridization of a labeled probe witha particular array member indicates that the sample from which the probewas derived expresses that gene. Differential gene expression analysisof disease tissue can provide valuable information. Microarraytechnology utilizes nucleic acid hybridization techniques and computingtechnology to evaluate the mRNA expression profile of thousands of geneswithin a single experiment. (see, e.g., WO 01/75166 published Oct. 11,2001; (See, for example, U.S. Pat. No. 5,700,637, U.S. Pat. No.5,445,934, and U.S. Pat. No. 5,807,522, Lockart, Nature Biotechnology,14:1675-1680 (1996); Cheung, V. G. et al., Nature Genetics21(Suppl):15-19 (1999) for a discussion of array fabrication). DNAmicroarrays are miniature arrays containing gene fragments that areeither synthesized directly onto or spotted onto glass or othersubstrates. Thousands of genes are usually represented in a singlearray. A typical microarray experiment involves the following steps: 1.preparation of fluorescently labeled target from RNA isolated from thesample, 2. hybridization of the labeled target to the microarray, 3.washing, staining, and scanning of the array, 4. analysis of the scannedimage and 5. generation of gene expression profiles. Currently two maintypes of DNA microarrays are being used: oligonucleotide (usually 25 to70 mers) arrays and gene expression arrays containing PCR productsprepared from cDNAs. In forming an array, oligonucleotides can be eitherprefabricated and spotted to the surface or directly synthesized on tothe surface (in situ).

The Affymetrix GeneChip® system is a commercially available microarraysystem which comprises arrays fabricated by direct synthesis ofoligonucleotides on a glass surface. Probe/Gene Arrays:Oligonucleotides, usually 25 mers, are directly synthesized onto a glasswafer by a combination of semiconductor-based photolithography and solidphase chemical synthesis technologies. Each array contains up to 400,000different oligos and each oligo is present in millions of copies. Sinceoligonucleotide probes are synthesized in known locations on the array,the hybridization patterns and signal intensities can be interpreted interms of gene identity and relative expression levels by the AffymetrixMicroarray Suite software. Each gene is represented on the array by aseries of different oligonucleotide probes. Each probe pair consists ofa perfect match oligonucleotide and a mismatch oligonucleotide. Theperfect match probe has a sequence exactly complimentary to theparticular gene and thus measures the expression of the gene. Themismatch probe differs from the perfect match probe by a single basesubstitution at the center base position, disturbing the binding of thetarget gene transcript. This helps to determine the background andnonspecific hybridization that contributes to the signal measured forthe perfect match oligo. The Microarray Suite software subtracts thehybridization intensities of the mismatch probes from those of theperfect match probes to determine the absolute or specific intensityvalue for each probe set. Probes are chosen based on current informationfrom GenBank and other nucleotide repositories. The sequences arebelieved to recognize unique regions of the 3′ end of the gene. AGeneChip Hybridization Oven (“rotisserie” oven) is used to carry out thehybridization of up to 64 arrays at one time. The fluidics stationperforms washing and staining of the probe arrays. It is completelyautomated and contains four modules, with each module holding one probearray. Each module is controlled independently through Microarray Suitesoftware using preprogrammed fluidics protocols. The scanner is aconfocal laser fluorescence scanner which measures fluorescenceintensity emitted by the labeled cRNA bound to the probe arrays. Thecomputer workstation with Microarray Suite software controls thefluidics station and the scanner. Microarray Suite software can controlup to eight fluidics stations using preprogrammed hybridization, wash,and stain protocols for the probe array. The software also acquires andconverts hybridization intensity data into a presence/absence call foreach gene using appropriate algorithms. Finally, the software detectschanges in gene expression between experiments by comparison analysisand formats the output into .txt files, which can be used with othersoftware programs for further data analysis.

In some embodiments, FGF19 gene deletion, gene mutation, or geneamplification is detected. Gene deletion, gene mutation, oramplification may be measured by any one of a wide variety of protocolsknown in the art, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, Proc.Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis),or in situ hybridization (e.g., FISH), using an appropriately labeledprobe, cytogenetic methods or comparative genomic hybridization (CGH)using an appropriately labeled probe. In addition, these methods may beemployed to detect FGF19 ligand gene deletion, ligand mutation, or geneamplification. As used herein, “detecting FGF19 expression” encompassesdetection of FGF19 gene deletion, gene mutation or gene amplification.

Additionally, one can examine the methylation status of the FGF 19 genein a tissue or cell sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells, and can result in alteredexpression of various genes. A variety of assays for examiningmethylation status of a gene are well known in the art. For example, onecan utilize, in Southern hybridization approaches, methylation-sensitiverestriction enzymes which cannot cleave sequences that containmethylated CpG sites to assess the methylation status of CpG islands. Inaddition, MSP (methylation specific PCR) can rapidly profile themethylation status of all the CpG sites present in a CpG island of agiven gene. This procedure involves initial modification of DNA bysodium bisulfite (which will convert all unmethylated cytosines touracil) followed by amplification using primers specific for methylatedversus unmethylated DNA. Protocols involving methylation interferencecan also be found for example in Current Protocols In Molecular Biology,Unit 12, Frederick M. Ausubel et al. eds., 1995; De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999); Brooks et al, Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536); and Lethe et al., Int. J. Cancer76(6): 903-908 (1998). As used herein, “detecting FGF19 expression”encompasses detection of FGF19 gene methylation.

In one aspect, the invention provides detection of expression of FGFR4polypeptide and/or polynucleotide (alone or in conjunction(simultaneously and/or sequentially)) with FGF19 expression) in abiological sample. Using methods known in the art, including thosedescribed herein, the polynucleotide and/or polypeptide expression ofFGFR4 can be detected. By way of example, the IHC techniques describedabove may be employed to detect the presence of one of more suchmolecules in the sample. As used herein, “in conjunction” is meant toencompass any simultaneous and/or sequential detection. Thus, it iscontemplated that in embodiments in which a biological sample is beingexamined not only for the presence of FGF19, but also for the presenceof FGFR4, separate slides may be prepared from the same tissue orsample, and each slide tested with a reagent that binds to FGF19 and/orFGFR4, respectively. Alternatively, a single slide may be prepared fromthe tissue or cell sample, and antibodies directed to FGF19 and FGFR4may be used in connection with a multi-color staining protocol to allowvisualization and detection of the FGF19 and FGFR4.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGFR4 expression and/or activity, the methodscomprising detecting FGFR4 in a biological sample from an individual. Insome embodiments, FGFR4 expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder, such as colorectal cancer, lung cancer,hepatocellular carcinoma, breast cancer and/or pancreatic cancer. Insome embodiment, the biological sample is serum or of a tumor.

In another aspect, the invention provides methods for diagnosing adisorder associated with FGFR4 and FGF19 expression and/or activity, themethods comprising detecting FGFR4 and FGF19 in a biological sample froman individual. In some embodiments, the FGF19 expression is increasedexpression or abnormal expression. In some embodiments, FGFR4 expressionis increased expression or abnormal expression. In some embodiments, thedisorder is a tumor, cancer, and/or a cell proliferative disorder, suchas colorectal cancer, lung cancer, hepatocellular carcinoma, breastcancer and/or pancreatic cancer. In some embodiment, the biologicalsample is serum or of a tumor. In some embodiments, expression of FGFR4is detected in a first biological sample, and expression of FGF19 isdetected in a second biological sample.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGFR4expression in an individual's biological sample, if any; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGFR4 expression detected instep (a). In some embodiments, increased FGFR4 expression in theindividual's biological sample relative to a reference value or controlsample is detected. In some embodiments, decreased FGFR4 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected in the individual. In some embodiments, FGFR4expression is detected and treatment with an anti-FGF19 antibody isselected.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) detecting FGF19and FGFR4 expression in the biological sample, if any; and (b)subsequence to step (a), selecting treatment for the individual, whereinthe selection of treatment is based on the FGF19 and FGFR4 expressiondetected in step (a). In some embodiments, increased FGF19 expression inthe individual's biological sample relative to a reference value orcontrol sample is detected. In some embodiments, decreased FGF19expression in the individual's biological sample relative to a referencevalue or control sample is detected in the individual. In someembodiments, increased FGFR4 expression in the individual's biologicalsample relative to a reference value or control sample is detected. Insome embodiments, decreased FGFR4 expression in the individual'sbiological sample relative to a reference value or control sample isdetected in the individual. In some embodiments, FGFR4 and FGF19expression are detected and treatment with an anti-FGF19 antibody isselected. In some embodiments, expression of FGFR4 is detected in afirst biological sample, and expression of FGF19 is detected in a secondbiological sample.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-FGF19 antibody, furtherwherein FGF19 expression and/or FGFR4 is detected in cells and/or tissuefrom the human patient before, during or after administration of ananti-FGF19 antibody. In some embodiments, FGF19 over-expression isdetected before, during and/or after administration of an anti-FGF19antibody. In some embodiments, FGFR4 expression is detected before,during and/or after administration of an anti-FGF19 antibody. Expressionmay be detected before; during; after; before and during; before andafter; during and after; or before, during and after administration ofan anti-FGF19 antibody.

In some embodiments involving detection, expression of FGFR4 downstreammolecular signaling is detected in addition to or as an alternative todetection of FGFR4 detection. In some embodiments, detection of FGFR4downstream molecular signaling comprises one or more of detection ofphosphorylation of MAPK, FRS2 or ERK2.

Some embodiments involving detection further comprise detection of Wntpathway activation. In some embodiments, detection of Wnt pathwayactivation comprises one or more of tyrosine phosphorylation ofβ-catenin, expression of Wnt target genes, β-catenin mutation, andE-cadherin binding to β-catenin. Detection of Wnt pathway activation isknown in the art, and some examples are described and exemplifiedherein.

In some embodiments, the treatment is for a cancer selected from thegroup consisting of colorectal cancer, lung cancer, ovarian cancer,pituitary cancer, pancreatic cancer, mammary fibroadenoma, prostatecancer, head and neck squamous cell carcinoma, soft tissue sarcoma,breast cancer, neuroblastomas, melanoma, breast carcinoma, gastriccancer, colorectal cancer (CRC), epithelial carcinomas, brain cancer,endometrial cancer, testis cancer, cholangiocarcinoma, gallbladdercarcinoma, and hepatocellular carcinoma.

Biological samples are described herein, e.g., in the definition ofBiological Sample. In some embodiment, the biological sample is serum orof a tumor.

In embodiments involving detection of FGF19 and/or FGFR4 expression,FGF19 and/or FGFR4 polynucleotide expression and/or FGF19 and/or FGFR4polypeptide expression may be detected. In some embodiments involvingdetection of FGF19 and/or FGFR4 expression, FGF19 and/or FGFR4 mRNAexpression is detected. In other embodiments, FGF19 and/or FGFR4polypeptide expression is detected using an anti-FGF19 agent and/or ananti-FGFR4 agent. In some embodiments, FGF19 and/or FGFR4 polypeptideexpression is detected using an antibody. Any suitable antibody may beused for detection and/or diagnosis, including monoclonal and/orpolyclonal antibodies, a human antibody, a chimeric antibody, anaffinity-matured antibody, a humanized antibody, and/or an antibodyfragment. In some embodiments, an anti-FGF19 antibody described hereinis use for detection. In some embodiments, FGF19 and/or FGFR4polypeptide expression is detected using immunohistochemistry (IHC). Insome embodiments, FGF19 expression is scored at 2 or higher using anIHC.

In some embodiments involving detection of FGF19 and/or FGFR4expression, presence and/or absence and/or level of FGF19 and/or FGFR4expression may be detected. FGF19 and/or FGFR4 expression may beincreased. It is understood that absence of FGF19 and/or FGFR4expression includes insignificant, or de minimus levels. In someembodiments, FGF19 expression in the test biological sample is higherthan that observed for a control biological sample (or control orreference level of expression). In some embodiments, FGF19 expression isat least about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 75-fold, 100-fold, 150-fold higher, or higher in the testbiological sample than in the control biological sample. In someembodiments, FGF19 polypeptide expression is determined in animmunohistochemistry (“IHC”) assay to score at least 2 or higher forstaining intensity. In some embodiments, FGF19 polypeptide expression isdetermined in an IHC assay to score at least 1 or higher, or at least 3or higher for staining intensity. In some embodiments, FGF19 expressionin the test biological sample is lower than that observed for a controlbiological sample (or control expression level).

In some embodiments, FGF19 expression is detected in serum and FGFR4expression is detected in a tumor sample. In some embodiments, FGF19expression and FGFR4 expression are detected in a tumor sample. In someembodiments, FGF19 expression is detected in serum or a tumor sample,and FGFR4 downstream molecular signaling and/or FGFR4 expression isdetected in a tumor sample. In some embodiments, FGF19 expression isdetected in serum or a tumor sample, and Wnt pathway activation isdetected in a tumor sample. In some embodiments, FGF19 expression isdetected in serum or a tumor sample, and FGFR4 downstream molecularsignaling and/or FGFR4 expression and/or Wnt pathway activation isdetected in a tumor sample.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition(s)effective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Moreover, the article of manufacture maycomprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody of the invention; and (b)a second container with a composition contained therein. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the first and second antibodycompositions can be used to treat a particular condition, e.g. cancer.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES

The following materials and methods were used in the Examples.

Residue numbers are according to Kabat (Kabat et al., Sequences ofproteins of immunological interest, 5th Ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)).

Direct Hypervariable Region Grafts onto the Acceptor Human ConsensusFramework

The phagemid used for this work is a monovalent Fab-g3 display vectorand consists of 2 open reading frames under control of a single phoApromoter. The first open reading frame consists of the stII signalsequence fused to the VL and CH1 domains of the acceptor light chain andthe second consists of the stII signal sequence fused to the VH and CH1domains of the acceptor heavy chain followed by the minor phage coatprotein P3.

To make the HVR grafts, hypervariable regions from murine 1A6 antibody(mu1A6) (FIG. 8; see co-owned U.S. patent application Ser. No.11/673,411, filed Feb. 9, 2007) were grafted into the huKI and huIIIconsensus acceptor frameworks to generate the direct HVR-graft of 1A6(1A6-graft) (FIGS. 1 and 2). In the VL domain the following regions weregrafted to the human consensus acceptor: positions 24-34 (L1), 50-56(L2) and 89-97 (L3). In the VH domain, positions 26-35 (H1), 49-65 (H2)and 93-102 (H3) were grafted. MacCallum et al. (MacCallum et al. J. Mol.Biol. 262: 732-745 (1996)) have analyzed antibody and antigen complexcrystal structures and found positions 49, 93 and 94 of the heavy chainare part of the contact region thus it seems reasonable to include thesepositions in the definition of HVR-H2 and HVR-H3 when humanizingantibodies. Correct clones were assessed by DNA sequencing.

Affinity Maturation

Human FGF19 was expressed in CHO cells and purified by conventionalmeans.

For affinity maturation, phage libraries based upon the HVR graft weregenerated that had mutations introduced in to the HVR loops, e.g., asdescribed in Dennis, WO2005080432.

High affinity clones were identified through five rounds of panningagainst human FGF19 protein with progressively increased stringency.Briefly, for the first 2 rounds of selection, FGF19 was immobilizeddirectly on MaxiSorp microtiter plates (Nunc) at 2 μg/ml in PBS.Successive rounds of selection used biotinylated-FGF19 (b-FGF19) in asoluble selection method (see, e.g., Fuh et al. J. Mol. Biol. (2004)).FGF19 was biotinylated (b-FGF-19) using Sulfo-NHS-LC-biotin (Pierce). Ashort binding period and low concentrations of b-FGF19 were utilized toenable selection of clones possessing faster association rates.

Fab and IgG Production

To express Fab protein for affinity measurements, a stop codon wasintroduced between the heavy chain and g3 in the phage display vector.Clones were transformed into E. coli 34B8 cells and grown in CompleteC.R.A.P. media at 30° C. (Presta et al. Cancer Res. 57: 4593-4599(1997)). Cells were harvested by centrifugation, suspended in PBS, 100uM PMSF, 100 uM benzamidine, 2.5 mM EDTA and broken open using amicrofluidizer. Fab was purified with Protein G affinity chromatography.

For screening purposes, IgG variants were initially produced in 293cells. Vectors coding for VL and VH (25 μg) were transfected into 293cells using the FuGene system. 500 uL of FuGENE was mixed with 4.5 mL ofDMEM media containing no FBS. This was incubated at room temperature for5 minutes. The 25 μg of each chain is added to this mixture andincubated at room temperature for 20 minutes. 1 mL of mixture waspipetted into each flask for transfection overnight at 37 C in 5% CO2.The following day the media containing the transfection mixture wasremoved and replaced with 23 mL PS04 media with 0.1 mL/L of traceelements (A0934) and 10 mg/L of insulin (A0940). Cells were returned tothe 37 C 5% CO2 incubator for an additional 5 days after which the mediawas harvested. The media was spun at 1000 rpm for 5 minutes and thensterile filtered using a 0.22 μm low protein binding filter. 2.5 mL of0.1 M PMSF was added for every 125 mL of media as a protease inhibitorand then stored at 4 C.

Affinity Determinations

Affinity determinations were performed by surface plasmon resonanceusing a BIAcore™-2000. Two protocols were used. Purified 1A6 variant IgGwas immobilized directly (approximately 550 RU) in 10 mM sodium acetatepH 4.8 on a CM5 sensor chip and serial 2-fold dilutions of the FGF19(0.08-1250 nM) in PBST were injected at a flow rate of 30 μl/min. Eachsample was analyzed with 4-minute association and 10-minutedissociation. After each injection the chip was regenerated using 10 mMGlycine pH 1.7. Binding response was corrected by subtracting the RUfrom a flow cell with an irrelevant IgG immobilized at similar density.A 1:1 Languir model of simultaneous fitting of k_(on) and k_(off) wasused for kinetics analysis.

Unpurified 1A6 variant IgG was also assayed from culture supernatantsusing an anti-human IgG capture method on the BIAcore™ 2000.Approximately 2700 RU of rabbit anti-human IgG (Pierce #31143) wasimmobilized in 10 mM sodium acetate pH 4.0 on a CM5 sensor chip. Theconcentration of unpurified 1A6 variant IgG was normalized to captureapproximately 200 RU of IgG from 5 μL of supernatant; an irrelevant IgGwas captured on a control flow cell. FGF19 (a 2-fold serial dilution,0.08 to 1000 nM in PBST) was injected at a flow rate of 30 μL/min. Eachsample was analyzed with 4-minute association and 10-minutedisassociation. After each injection the chip was regenerated using 10mM Glycine pH 1.7. The immobilized anti-human IgG was then rechargedwith culture supernatant containing unpurified 1A6 variant IgG for thenext dilution of FGF19. Binding response was corrected by subtractingthe irrelevant IgG flow cell control from 1A6 variant IgG flow cells. A1:1 Languir model of simultaneous fitting of k_(on) and k_(off) was usedfor kinetics analysis.

Solid Phase Receptor Binding Assay

Maxisorb 96 well plates were coated overnight at 4° C. with 50 μl of 2μg/ml anti-human immunoglobulin Fcγ fragment specific (JacksonImmunoresearch) and used to capture 1 μg/ml FGFR-Fc chimeric proteins (R& D Systems). The non-specific binding sites were saturated with PBS/3%BSA for 1 hour and FGF19 (0.25 μg/ml) was incubated for 2 h in PBS/0.3%BSA in the presence of oligosaccharides (0.5 μg/ml; Neoparin Inc.) andthe indicated anti-FGF19 antibody (0-10 μg/ml). FGF19 binding wasdetected using a biotinylated FGF19 specific polyclonal antibody (0.5μg/ml; BAF969; R & D Systems) followed by streptavidin-HRP and TMBcolorimetric substrate.

FGFR4/MAPK Phosphorylation

HEPG2 cells starved overnight in serum free media were treated with 250ng/ml FGF19 for 10 min in the presence or the absence of antibodies.Cells were lysed in R27A buffer (Upstate) with 10 mM NaF, 1 mM sodiumorthovanadate, and complete protease inhibitor tablet (Roche). Lysateswere prepared, electrophoresed and analyzed by Immunoblot usinganti-phospho-MAPK and anti-MAPK specific antibodies (Cell Signaling).For immunoprecipitation of FGFR4, equal amounts of proteins wereincubated with 1 μg specific anti-FGFR4 (1G7; Genentech, Inc.) antibodyimmobilized onto protein A-Sepharose for 2 h at 4° C. then washed withlysis buffer and eluted with 2× Laemmli buffer, boiled, andmicrocentrifuged. Immunoblotting was performed with anti-phosphotyrosineantibody (4G10, UpState), anti-phospho-ERK2 antibody (Santa CruzBiotech). Membranes were stripped (Pierce) and reprobed with appropriateantibodies to determine total proteins.

Western Blot for FGF19

Liver tissues were homogenized in modified RIPA buffer (50 mM Tris-Cl,pH 7.5; 150 mM NaCl; 1% IGEPAL; 1 mM EDTA; 0.25% sodium deoxycholate; 1mM NaF; 1 mM Na3VO4; protease inhibitors cocktail (Sigma-Aldrich, St.Louis, Mo.) and clarified by centrifugation. Protein concentrations ofthe lysates were determined using the BCA protein assay reagent (Pierce,Rockford, Ill.). Equal amounts of proteins were incubated with specificantibody immobilized onto protein A-Sepharose (Sigma-Aldrich) for 2hours at 4° C. with gentle rotation. Beads were washed extensively withlysis buffer and immunecomplexes were eluted in 2× Laemmli buffer,boiled and microcentrifuged. Proteins were resolved on SDS-PAGE,transferred to nitrocellulose membrane and incubated with specificprimary antibodies. After washing and incubating with secondaryantibodies, immunoreactive proteins were visualized by the ECL detectionsystem (Amersham, Arlington Ht. IL). Recombinant human and cynomolgusproteins were loaded at a concentration of 100 ng or 200 ng.

Xenograft Experiment

Six- to eight-week-old athymic BALB/c female mice (Charles Rivers Inc.)were inoculated subcutaneously with 5×10⁶ HCT116 colon tumor (200μl/mouse). After 7 days, mice bearing tumors of equivalent volumes (˜100mm³) were randomized into groups (n=10) and treated intraperitoneallyonce weekly. Tumors were measured with an electronic caliper (FowlerSylvac Ultra-Cal Mark III) and average tumor volume was calculated usingthe formula: (W2×L)/2 (W, the smaller diameter; L, the larger diameter).

FGFR4, FRS2, ERK and β-Catenin Phosphorylation in Xenograft Tumors

Tumors excised from treated animals were homogenized in lysis buffer [50mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% sodiumdeoxycholate, 1 mM NaF, 1 mM sodium orthovanadate and complete proteaseinhibitor (Roche)]. Equal amounts of proteins were incubated with 1 μgspecific FGFR4 (1G7; Genentech, Inc.) or FRS2 (UpState) antibodyimmobilized onto protein A-Sepharose for 2 h at 4° C. then washed withlysis buffer and eluted with 2× Laemmli buffer, boiled, andmicrocentrifuged. Immunoblots were done with anti-phosphotyrosineantibody (4G10, UpState), anti-phospho-ERK2 antibody (Santa CruzBiotech) or anti-N-terminally dephosphorylated β-catenin antibody(UpState). Membranes were stripped (Pierce) and reprobed withappropriate antibodies to determine total proteins.

Results and Discussion

Humanization of 1A6

The human acceptor framework used for humanization of 1A6 consists ofthe consensus human kappa I VL domain and the human subgroup IIIconsensus VH domain. The VL and VH domains of mu1A6 were aligned withthe human kappa I and subgroup III domains; each HVR was identified andgrafted into the human acceptor framework to generate a 1A6 HVR graftthat could be displayed as an Fab on phage (FIGS. 1 and 2).

Phage expressing the 1A6-graft bound to immobilized huFGF19; however,when 1A6-graft was expressed as an IgG, Biacore analysis of its affinityfor FGF19 revealed that binding affinity had been reduced by over50-fold relative to the chimeric 1A6 antibody, largely due to areduction in the association rate (K_(on)) (Table 2).

TABLE 2 Biacore analysis of chimeric 1A6 and 1A6-graft Binding tosoluble Human FGF19 Ka (M/s) Kd (s−1) KD (pM) chimeric 1A6 1.47E+065.30E−05 36 1A6-graft 5.93E+04 1.24E−04 2091

Phage libraries based upon the 1A6 HVR graft were generated that hadmutations introduced in to the HVR loops. These libraries were pannedfor 2 rounds against immobilized FGF19 followed by 3 additional roundsof selection using short durations for binding to low concentrations ofsoluble b-FGF19. Enrichment, defined as the number of phage recovered inthe presence of b-FGF divided by the number of phage recovered in theabsence of b-FGF, was observed beginning after round 3. Following 5rounds of selection, clones were picked for DNA sequence analysis.Sequence changes targeting each of the HVRs were observed (FIG. 3).

Selected clones were reformatted as IgG for further analysis by Biacore.Several clones had improved affinities compared to the 1A6-graftantibody (Table 3). These clones had changes in the light chain variableregion (S34T, S34A or Q90S) or in the heavy chain variable region V34A,H35Q, V50L, A100bR or A100bP/M100cS).

TABLE 3 Biacore analysis of selected affinity matured antibodies Bindingto soluble Human FGF19 Ka (fold Kd (fold KD (fold slower) faster)weaker) chimeric 1A6 1 1 1 1A6- graft 24.8 2.3 58.1 hu1A6.S34T (HVR-L1)12.0 1.2 14.0 hu1A6.S34A (HVR-L1) 7.7 1.2 9.1 hu1A6.Q90S (HVR-L3) 10.41.5 16.0 hu1A6.V34A (HVR-H1) 12.5 0.9 10.5 hu1A6.H35Q (HVR-H1) 5.0 1.36.6 hu1A6.V50L (HVR-H2) 6.3 0.9 5.7 hu1A6.A100bR (HVR-H3) 2.6 0.9 2.3hu1A6.A100bP/M100kS (HVR-H3) 1.7 0.5 0.9

The best clones had 1 change from 1A6-graft (either S34A or S34G) andshowed similar binding affinity to the murine 1A6 antibody for humanFGF19.

Elimination of a Potential Iso-Aspartic Acid Forming Site in HVR-L2 ofHumanized 1A6

To avoid potential manufacturing issues, a potential iso-aspartic acidforming site (Asp-Gly) in HVR-L2 of the humanized 1A6 variants waseliminated by converting D56 either to Glu (D56E) or Ser (D56S). Neithersubstitution had an effect on binding FGF19 as determined by Biacore.Tables 4 and 5 show the Biacore analysis of the D56S substitutedantibodies.

TABLE 4 Biacore analysis of chimeric 1A6 antibody and affinity matured1A6 variants to human FGF19 Binding to soluble Human FGF19 Ka (M/s) Kd(s−1) KD (pM) chimeric 1A6 1.70E+06 5.40E−05 32 hu1A6.S34A/D56S 3.90E+054.60E−05 118 (HVR-L1/L2) hu1A6.S34G/D56S (HVR-L1/L2) 1.40E+05 1.60E−05114

TABLE 5 Biacore analysis of chimeric 1A6 antibody and affinity matured1A6 variants to cynomolgus FGF19 Binding to soluble Cyno FGF19 Ka (M/s)Kd (s−1) KD (pM) chimeric 1A6 7.60E+05 6.60E−05 87 hu1A6.S34A/D56S1.60E+05 7.70E−05 481 (HVR-L1/L2) hu1A6.S34G/D56S (HVR-L1/L2) 5.30E+044.80E−05 906

Thus, starting from a graft of the 6 murine 1A6 HVRs, the expansion ofHVR-H2 to include position 49 (Glycine), the expansion of HCR-H3 toinclude positions 93 (Valine) and 94 (Arginine), the addition of 1change in HVR-L1 leads to a fully humanized, high affinity 1A6 antibodywith a binding affinity for human FGF19 that is similar to that of theparent murine 1A6 antibody. Other humanized 1A6 variants have also beenidentified that are potentially therapeutically suitable. Furthermore,selected humanized antibodies described herein have been determined tohave at least comparable biological activity as the parent 1A6 antibody,for example in receptor phosphorylation assays, etc.

Characterization of an Antibody of the Invention

Humanized anti-FGF19 antibody 1A6.v1 was characterized as follows:

-   -   (1) In an assay to test ability of 1A6.v1 to block binding of        FGF19 to its receptor, FGFR4, 1A6.v1 was able to block FGF19        binding to its receptor at least as well as one comparator        antibody—namely a chimeric antibody (which comprised the        variable regions from the murine parent 1A6 antibody (variable        domains depicted in FIG. 8) fused to a human Fc region). When        tested across an antibody concentration range of about 1-67 nM,        under conditions as described in the Materials and Methods        section above, 1A6.v1 was found to have an IC50 value that was        similar to a comparator antibody such as the chimeric 1A6        antibody. See FIG. 9.    -   (2) 1A6.v1 was also tested for cross-species binding among human        and primate (Cynomolgus macaque monkey). 1A6.v1 was found to        bind specifically to human and primate (Cynomolgus monkey) FGF        19 receptor. In situ analysis revealed that cyno FGF19        expression in liver showed a similar pattern to human FGF19        expression in liver tissue. See FIG. 10.    -   (3) 1A6.v1 was tested for in vitro efficacy using a colon tumor        cell line (HCT116 cells). Results from this study showed that        the 1A6.v1 antibody was capable of inhibiting the        phosphorylation of FGFR4, FRS2 and ERK in vitro. See FIG. 11.    -   (4) 1A6.v1 was tested for in vivo efficacy using a tumor        xenograft model based on a colon tumor cell line (HCT116 cells).        Results from this efficacy study showed that the 1A6.v1 antibody        was capable of inhibiting growth of tumors in vivo. Moreover,        the phosphorylation of FGFR4, FRS2, and ERK was inhibited in        humanized anti-FGF19 antibody 1A6.v1-treated HCT116 xenograft        tumors. See FIG. 12.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

1. An isolated anti-FGF19 antibody comprising: (a) a light chaincomprising: (i) hypervariable region (HVR)-L1 comprising sequenceA1-A11, wherein A1-A11 is KASQDINSFLA (SEQ ID NO:11); (ii) HVR-L2comprising sequence B1-B7, wherein B1-B7 is RANRLVD (SEQ ID NO:2),RANRLVS (SEQ ID NO:13), or RANRLVE (SEQ ID NO:14); and (iii) HVR-L3comprising sequence C1-C9, wherein C1-C9 is LQYDEFPLT (SEQ ID NO:3); and(b) a heavy chain comprising: (i) HVR-H1 comprising sequence D1-D10,wherein D1-D10 is GFSLTTYGVH (SEQ ID NO:4); (ii) HVR-H2 comprisingsequence E1-E17, wherein E1-E17 is GVIWPGGGTDYNAAFIS (SEQ ID NO:7); and(iii) HVR-H3 comprising sequence F1-F13, wherein F1-F13 is VRKEYANLYAMDY(SEQ ID NO:8).
 2. The antibody of claim 1, wherein the antibody ishumanized.
 3. The antibody of claim 1, wherein at least a portion of theframework sequence is a human consensus framework sequence.
 4. Theantibody of claim 1, wherein B1-B7 is RANRLVD (SEQ ID NO:2).
 5. Theantibody of claim 1, wherein B1-B7 is RANRLVS (SEQ ID NO:13).
 6. Theantibody of claim 1, wherein B1-B7 is RANRLVE (SEQ ID NO:14).
 7. Theantibody of claim 1 comprising human K subgroup 1 consensus frameworksequence.
 8. The antibody of claim 1 comprising heavy chain humansubgroup III consensus framework sequence.
 9. Isolated nucleic acidencoding the antibody of claim
 1. 10. A host cell comprising the nucleicacid of claim
 9. 11. A pharmaceutical composition comprising theantibody of claim 1 and a pharmaceutically acceptable carrier.
 12. Amethod for making an anti-FGF19 antibody, said method comprisingculturing the host cell of claim 10 so that the antibody is produced.13. The method of claim 12, further comprising recovering the antibodyfrom the host cell.
 14. The method of claim 12, wherein the host cell iseukaryotic.